Advances in applied biotechnology proceedings of the 2nd international conference on applied biotechnology

Lecture Notes in Electrical Engineering 332

Tong-Cun Zhang
Motowo Nakajima
Editors

Advances
in Applied
Biotechnology
Proceedings of the 2nd International
Conference on Applied Biotechnology
(ICAB 2014)-Volume I

Tai Lieu Chat Luong


Lecture Notes in Electrical Engineering
Volume 332

Board of Series editors
Leopoldo Angrisani, Napoli, Italy
Marco Arteaga, Coyoacán, México
Samarjit Chakraborty, München, Germany
Jiming Chen, Hangzhou, P.R. China
Tan Kay Chen, Singapore, Singapore
Rüdiger Dillmann, Karlsruhe, Germany
Haibin Duan, Beijing, China
Gianluigi Ferrari, Parma, Italy
Manuel Ferre, Madrid, Spain
Sandra Hirche, München, Germany
Faryar Jabbari, Irvine, USA

Janusz Kacprzyk, Warsaw, Poland
Alaa Khamis, New Cairo City, Egypt
Torsten Kroeger, Stanford, USA
Tan Cher Ming, Singapore, Singapore
Wolfgang Minker, Ulm, Germany
Pradeep Misra, Dayton, USA
Sebastian Möller, Berlin, Germany
Subhas Mukhopadyay, Palmerston, New Zealand
Cun-Zheng Ning, Tempe, USA
Toyoaki Nishida, Sakyo-ku, Japan
Bijaya Ketan Panigrahi, New Delhi, India
Federica Pascucci, Roma, Italy
Tariq Samad, Minneapolis, USA
Gan Woon Seng, Nanyang Avenue, Singapore
Germano Veiga, Porto, Portugal
Haitao Wu, Beijing, China
Junjie James Zhang, Charlotte, USA


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Tong-Cun Zhang Motowo Nakajima
•

Editors

Advances in Applied
Biotechnology
Proceedings of the 2nd International
Conference on Applied Biotechnology
(ICAB 2014)-Volume I

123

Editors
Tong-Cun Zhang
Tianjin University of Science
and Technology
Tianjin
China

Motowo Nakajima
SBI ALApromo
Tokyo
Japan

ISSN 1876-1100
ISSN 1876-1119 (electronic)
Lecture Notes in Electrical Engineering
ISBN 978-3-662-45656-9
ISBN 978-3-662-45657-6 (eBook)
DOI 10.1007/978-3-662-45657-6
Library of Congress Control Number: 2014955624
Springer Heidelberg New York Dordrecht London
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Preface

The 2014 International Conference on Applied Biotechnology (ICAB 2014),
organized by Chinese Society of Biotechnology and Tianjin University of Science,
was held from November 28 to 30, 2014 in Tianjin, China.
The conference served as a forum for exchange and dissemination of ideas and
the latest findings in aspects of applied biotechnology. The conference was complemented by talks given by more than 30 professors and researchers.
The conference papers were submitted by more than 100 authors from different
universities, institutes and companies. Numerous fields were covered, ranging from
fermentation engineering, cell engineering, genetic engineering, enzyme engineering to protein engineering.
Special thanks are given to Secretary Staff of the conference for the commitment
to the conference organization. We would also like to thank all the authors who
contributed with their papers to the success of the conference.
This book gathers a selection of the papers presented at the conference. It
contains contributions from both academic and industrial researchers focusing on
the research and development of applied biotechnology from all over the world.
The scientific value of the papers also helps researchers in this field to get more
valuable results.
Tianjin, China
Tokyo, Japan

Tong-Cun Zhang
Motowo Nakajima

v


Contents

Part I
1

2

3

4

5

6

Microbial Genetics and Breeding

Cloning and Bioinformatics Analysis of spsC Gene
from Sphingomonas sanxanigenens NX02. . . . . . . . . . . . . . . . . . .
Xiaoyan Li, Haidong Huang, Mingming Zhou and Peng Zhang

3

Preliminary Study on Salt Resistance Seedling Trait

in Maize by SRAP Molecular Markers . . . . . . . . . . . . . . . . . . . .
Chunyang Xiang, Jin Du, Peipei Zhang, Gaoyi Cao and Dan Wang

11

Isolation of Differentially Expressed Genes from Groundnut
Genotypes Differing in Seed Dormancy . . . . . . . . . . . . . . . . . . . .
Bo Qu, Yue Yi Tang, Xiu Zhen Wang, Qi Wu, Quan Xi Sun,
Shu Yan Guan, Chuan Tang Wang and Pi Wu Wang
Increase of the Lycopene Production in the Recombinant
Strains of Escherichia coli by Supplementing with Fructose. . . . . .
Tong-Cun Zhang, Wen Li, Xue-Gang Luo, Cui-Xia Feng,
Ming-Hui Zhang, Wen Du and De-Yun Ma
Isolation of Differentially Expressed Genes from Developing
Seeds of a High-Protein Peanut Mutant and Its Wild Type
Using GenefishingTM Technology . . . . . . . . . . . . . . . . . . . . . . . . .
Shu Tao Yu, Hong Bo Yu, Guo Qing Yu, Li Ren Zhao,
Hong Xi Sun, Yue Yi Tang, Xiu Zhen Wang, Qi Wu,
Quan Xi Sun and Chuan Tang Wang
Identification of the Binding Domains of Nedd4 E3 Ubiquitin
Ligase with Its Substrate Protein TMEPAI . . . . . . . . . . . . . . . . .
Lei Jing, Xin Huo, Yufeng Li, Yuyin Li and Aipo Diao

19

29

37

47

vii


viii

7

8

9

10

11

12

13

14

15

Contents

Optimization of the Fermentation Conditions of Pep-1-Fused
EGF in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tong-Cun Zhang, De-Yun Ma, Xue-Gang Luo and Yue Wang

55

Characterization of Rhamnolipid Production
in a Pseudomonas aeruginosa Strain . . . . . . . . . . . . . . . . . . . . . .
Cuikun Zhang and Hongjiang Yang

61

High-Quality Protein-Encoding Gene Design and Protein
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guo-qing Huang, Lei Wang, Dong-kai Wang, Qiong Wu,
Yao Li, Jin-hai Zhao and Di-fei Cao
Isolation and Characterization of a Highly Siderophore
Producing Bacillus subtilis Strain. . . . . . . . . . . . . . . . . . . . . . . . .
Huiming Zhu and Hongjiang Yang
Isolation and Identification of an Inulinase-Producing Strain
and the Optimization of Its Fermentation Condition. . . . . . . . . . .
Yang Zhang, Hongyang Zhu, Jinhai Wang, Xiuling Zhou,
Wei Xu and Haiying Shi
Isolation and Identification of a Cellulose-Producing
Bacterial Strain from the Genus Bacillus . . . . . . . . . . . . . . . . . . .
Hongyang Zhu, Yang Zhang, Jinhai Wang,
Yongning Li and Weiling Lin
Improved Lactose Utilization by Overexpression
β-Galactosidase and Lactose Permease
in Klebsiella pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Xuewu Guo, Yazhou Wang, Xiangyu Guan, Yefu Chen,
Cuiying Zhang and Dongguang Xiao
Breeding High Producers of Enduracidin
from Streptomyces fungicidicus by Combination

of Various Mutation Treatments . . . . . . . . . . . . . . . . . . . . . . . . .
Dong Zhang, Qingling Wang and Xinle Liang
Expression of Stichopus japonicus Lysozyme Gene
in Bacillus subtilis WB600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhiwen Liu, Xingyu Liao, Lu Sun, Dan Zou,
Dan Li and Lina Cong

73

83

93

109

121

133

143


Contents

ix

16

Mega-Genome DNA Extraction from Pit Mud . . . . . . . . . . . . . . .
Huimin Xie, Yali Dai and Lin Yuan

155

17

Evidence for a Link of SDPR and Cytoskeleton . . . . . . . . . . . . . .
Baoxia Zhang, Jun Zhu, Liqiao Ma, Yuyin Li,
Aipo Diao and Yinchuan Li

165

18

CREB Regulated Transcription Coactivator 1 (CRTC1)
Interacts with Microtubules. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Liqiao Ma, Yu Sun, Baoxia Zhang, Yuyin Li,
Aipo Diao and Yinchuan Li

19

20

The Biological Effects of Carbon Nanotubes in Plasma
Membranes Damage, DNA Damage, and Mitochondrial
Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhuo Zhao, Zhi-Peng Liu, Hua Wang, Feng-Juan Liu, Hui Zhang,
Cong-Hui Zhang, Chen-Guang Wang and Xiao-Chuan Jia
Evidence of the Interplay of Menin, CRTC1 and THOC5
Triangles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lichang Wu, Qiwen Zhang, Liqiao Ma, Yu Sun, Baoxia Zhang,

Caicai Kang, Aipo Diao and Yinchuan Li

Part II
21

22

23

24

173

179

189

Optimization and Control of Biological Process

Classification of Lymphoma Cell Image Based
on Improved SVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ting Yan, Quan Liu, Qin Wei, Fen Chen and Ting Deng

199

Foam Control in Epothilones Fermentation of
Sorangium cellulosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yue Liu, Lin Zhao, Hongrui Zhang, Fuming He and Xinli Liu

209

Acute Toxicity by Four Kinds of Oil Dispersants
in Cynoglossus semilaevis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jinwei Gao, Wenli Zhou and Ruinan Chen

219

Imprinted Cross-Linked Enzyme Aggregate (iCLEA)
of Phenylalanine Ammonia Lyase: A New Stable Biocatalyst . . . .
Jian Dong Cui, Rong Lin Liu and Lian Lian Li

223


x

25

26

27

28

29

30

31

32

33

34

Contents

Effects of Calcium on the Morphology of Rhizopus oryzae
and L-lactic Acid Production. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yong-Qian Fu, Long-Fei Yin, Ru Jiang, Hua-Yue Zhu
and Qing-Cheng Ruan
Estimation of Dietary Copper (Cu) Requirement
of Cynoglossus semilaevis Günther . . . . . . . . . . . . . . . . . . . . . . . .
Qingkui Wang, Yang Zhang, Dongqing Bai, Chengxun Chen,
Yongjun Guo and Kezhi Xing
Influence of Different Substrates on the Production
of Pigments and Citrinin by Monascus FJ46 . . . . . . . . . . . . . . . .
Hongxia Mu, Liubin Huang, Xuemei Ding and Shuxin Zhao
FAD2B from a Peanut Mutant with High Oleic Acid
Content Was Not Completely Dysfunctional. . . . . . . . . . . . . . . . .
Xiu Zhen Wang, Qi Wu, Yue Yi Tang, Quan Xi Sun
and Chuan Tang Wang

233

245

257

265

Optimization of Sterilization Process After Spore Activation
for Cereal Beverage in Large-Scale Production . . . . . . . . . . . . . .
Zhe Li, Liping Zhu, Shigan Yan, Junjie Liu and Wenjuan Zhao

273

Optimization of Medium for Exopolysaccharide Production
by Agaricus brunnescens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Li-tong Ban, Yu Wang, Liang Huang and Hongpeng Yang

283

Effect of Attapulgite on Cell Activity
of Steroid-Transforming Arthrobacter simplex . . . . . . . . . . . . . . .
Yanbing Shen, Hengsheng Zhao, Yanhua Liu,
Rui Tang and Min Wang
Establishment of a Method to Measure the Interaction
Between Nedd4 and UbCH5c for Drug Screening. . . . . . . . . . . . .
Kunyuan Kou, Jianli Dang, Baoxia Zhang, Guanrong Wu,
Yuyin Li and Aipo Diao
Determination of Phthalate Esters in Tea
by Gas Chromatography–Mass Spectrometry . . . . . . . . . . . . . . .
Yan Lu, Liping Du, Yang Qiao, Tianlu Wang
and Dongguang Xiao
Antibacterial Mechanism of 10-HDA Against Bacillus subtilis . . . .
Xiaohui Yang, Junlin Li and Ruiming Wang

289

297

305

317


Contents

35

36

37

38

39

40

41

42

Monitoring Glutamate and Glucose Concentration During
the Temperature Triggered Glutamate Fermentation
by Near-Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . .
Yongli Gui, Jingbo Liang, Chenglin Zhang, Xixian Xie,

Qingyang Xu, Ning Chen and Lei Ma
Effect of Sodium Citrate on L-tryptophan Fermentation
by Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Qing-yang Xu, Li-kun Cheng, Xi-xian Xie, Cheng-lin Zhang,
Yan-jun Li and Chen Ning
Reduction Reaction of Methyl Condensation Compound
by Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lu Yu, Shuhong Mao, Shaoxian Ji, Xiaoguang Liu
and Fuping Lu
Study on Ultrasonic-Assisted Extraction of Essential Oil
from Cinnamon Bark and Preliminary Investigation
of Its Antibacterial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ping Li, Lin Tian and Tao Li
Geranyl Butyrate Production by Candida antarctica
Lipase B-Displaying Pichia pastoris . . . . . . . . . . . . . . . . . . . . . . .
Zi Jin, Janvier Ntwali, Ying Lin, Huang Kui,
Suiping Zheng and Shuangyan Han

xi

325

335

343

349

361

Metabolic Analysis of a Corynebacterium glutamicum IdhA
Mutant During an Efficient Succinate Production Using
pH-Control Under Oxygen Deprivation . . . . . . . . . . . . . . . . . . . .
Chen Wang, Heng Cai, Zhihui Zhou, Hong-gui Wan
and Ping-kai Ouyang

375

Effects of Stimulators on Lutein and Chlorophyll Biosyntheses
in the Green Alga Chlorella pyrenoidosa Under Heterotrophic
Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tao Li, Dongqing Bai, Lin Tian, Ping Li, Yihan Liu
and Yue Jiang

389

Pharmacophore-Based Virtual Screening and Result Analysis
of Histone Methyltransferase SMYD3 Inhibitors . . . . . . . . . . . . .
Shaodan Liu, Ziyue Sun, Yonghui Zhong, Qingxin Cui,
Xuegang Luo and Yujie Dai

399


xii

43

44

45

46

Contents

Effects of K2HPO4 on the Growth of Nostoc Flagelliforme
in Liquid Media with Different Carbon Sources . . . . . . . . . . . . . .
Hexin Lv, Feng Xia, Shiru Jia, Xianggan Cui and Nannan Yuan

407

Production of Alkyl Polyglucoside Using Pichia pastoris
GS115 Displaying Aspergillus aculeatus β-Glucosidase I . . . . . . . .
Yajun Kang, Binru Wei, Dongheng Guo and Suiping Zheng

417

Enhancement of Gellan Production in Sphingomonas
paucimobilis JLJ by Heterogeneous Expression
of Vitreoscilla Hemoglobin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Qinglong Ji, Dan Li, Xiangqian Li, Ting Li and Lin Yuan
Enhanced Adenosine Production by Bacillus subtilis
at Condition with Comprehensively Controlled Dissolved
Oxygen and pH During Fermentation . . . . . . . . . . . . . . . . . . . . .
Yue Liu, Juhua He, Qingyang Xu, Chenglin Zhang,
Ning Chen and Xixian Xie

47

The Semi-continuous Cultivation of Nostoc flagelliforme Cells . . . .
Lifang Yue, Yupeng Xiao, Guojuan Sun, Shiru Jia,
Yujie Dai and Xing Zheng

48

Study on Ecological Diversity of Pectase and Its Producing
Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jing Xiao, Xiwang Zhou, Xiaolong Zhang and Ruiming Wang

Part III
49

50

51

427

439

453

461

Biological Separation and Biological Purification

The Extraction and Regeneration of Resin XAD-16
in the Purification of Epothilones. . . . . . . . . . . . . . . . . . . . . . . . .
Can Li, Lin Zhao, Xiaona Wang, Qiang Ren and Xinli Liu

Conversion Process of High Color Value Gardenia
Red Pigment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shangling Fang, Wei Jiang, Jinghua Cao, Xu Xu,
Yanyan Jing and Maobin Chen
Efficient Purification and Active Configuration Investigation
of R-phycocyanin from Polysiphonia urceolata . . . . . . . . . . . . . . .
Li-ping Zhu, Shi-gan Yan and Ai-jie Lv

469

479

489


Contents

52

53

54

55

56

57

58

59

60

Concentration of Sinigrin from Indian Mustard
(Brassica juncea L.) Seeds Using Nanofiltration Membrane. . . . . .
Tianxin Wang, Hao Liang and Qipeng Yuan
Optimization of Crude Polysaccharides Extraction
from Dioscorea esculenta by Response Surface Methodology. . . . .
Kaihua Zhang, Liming Zhang, Na Liu, Jianheng Song
and Shuang Zhang
Nanofiltration Extraction and Purification Method
for Cyclic Adenosine Monophosphate (cAMP)
from Chinese Date Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chunxia Wang, Yihan Liu, Hongbin Wang, Lianxiang Du
and Fuping Lu
Effect of Manchurian Walnut Extracts on Cancer Cells
Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Changcai Zhao, Xing Niu, Rui Huang, Jiali Dong,
Yuyin Li and Aipo Diao

xiii

497

509

521

533

Extraction and Purification of Lumbrokinase
from the “Ohira the 2nd” Earthworm . . . . . . . . . . . . . . . . . . . . .
Tianjun Li, Jian Ren, Tao Li and Yingchao Wang

541

Study on Green Crystallization Process for L-glutamic
Acid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhi-hua Li, Cheng-lin Zhang and Qing-yang Xu

547

Synthesis of Poly (β-L-malic Acid) by the Optimization
of Inorganic Nitrogen Complexing with Growth
Factors Using Aureobasidium pullulans CGMCC3337. . . . . . . . . .
Changsheng Qiao, Yumin Song, Zhida Zheng, Xujia Fan
and Shiyun Yu

557

Primary Characterization and In Vitro Antioxidant
Activities of Polysaccharides from Yam Peel . . . . . . . . . . . . . . . .
Na Liu, Liming Zhang, Kaihua Zhang, Aiying Tian
and Ruichao Li
Optimization of Sample Preparation for the Metabolomics
of Bacillus licheniformis by GC-MS . . . . . . . . . . . . . . . . . . . . . . .
Hongbin Wang, Zhixin Chen, Jihan Yang, Yihan Liu
and Fuping Lu

567

579


xiv

61

62

63

Contents

Characterization of Recombinant L-Amino Acid Deaminase
of Proteus mirabilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chenglin Zhang, Jia Feng, Xixian Xie, Qingyang Xu
and Ning Chen
Screening for Strains Capable of 13β-ethyl-4-gonene-3,
17-dione Biotransformation and Identification of Product . . . . . . .
Linlin Huang, Xiaoguang Liu, Yulan He, Pingping Wei,
Shuhong Mao and Fuping Lu
Extent and Pattern of DNA Cytosine Methylation Changes
Between Non-pollinated and Pollinated Ovaries
from Cymbidium hybridium . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Xiaoqiang Chen, Xiulan Li, Ning Sun and Wenqin Song

Part IV

589

597

607

Progress of Biotechnology

64

The Application Status of Microbes in Salted Fish Processing. . . .
Yan Yan Wu, Gang You and Lai Hao Li

65

Construction and Functional Analysis of Luciferase
Reporter Plasmids Containing ATM and ATR Gene
Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Li Zheng, Xing-Hua Liao, Nan Wang, Hao Zhou,
Wen-Jian Ma and Tong-Cun Zhang

619

627


Part I

Microbial Genetics and Breeding

Chapter 1

Cloning and Bioinformatics Analysis
of spsC Gene from Sphingomonas
sanxanigenens NX02
Xiaoyan Li, Haidong Huang, Mingming Zhou and Peng Zhang

Abstract Sphingomonas sanxanigenens strain NX02 synthesizes a novel sphingan
Ss, which can be used as drilling mud and thickening agent in the recovery of
petroleum by water flooding. In order to research genes involved in the biosyntheses of sphingan Ss, strain NX02 was induced by transposon mini-Tn5 on suicide
plasmid pUT, and a mutant strain T163, which cannot produce sphingan Ss, was
screened. The spsC gene of NX02 was obtained by the method of Tn5 flanking
PCR and LP-RAPD. The predicted amino acid sequence of the spsC protein possessed 493 amino acids and a calculated molecular mass of 53.5 kDa. Bioinformatics analysis revealed that spsC had the highest 60 % amino acid sequence
identity with polysaccharide biosynthesis protein of Novosphingobium lindaniclasticum LE124. spsC protein had typical polysaccharide polymerases family
transmembrane helices, located between amino acids Y13-V44 and P411-L436.
The N-terminal sequence of spsC had high identity to chain length determinant
protein of Wzz superfamily.
Keywords Sphingomonas sanxanigenens
Bioinformatics




Polysaccharide




Sphingan Ss

X. Li
College of Food Science and Bioengineering, Tianjin Agricultural University,
Tianjin 300384, China
e-mail:
H. Huang (&)
M. Zhou
P. Zhang
College of Agronomy and Environmental Resources, Tianjin Agricultural University,
Tianjin 300384, China
e-mail:
© Springer-Verlag Berlin Heidelberg 2015
T.-C. Zhang and M. Nakajima (eds.), Advances in Applied Biotechnology,
Lecture Notes in Electrical Engineering 332, DOI 10.1007/978-3-662-45657-6_1

3


4

X. Li et al.

1.1 Introduction
A number of bacteria of the genus Sphingomonas produce polysaccharides called
sphingans, including gellan, welan, S-88, rhamsan, and diutan [1–3]. Sphingans
share the similar tetrasaccharide backbone structures and divergent side chains.
Because of their excellent rheological characteristics, sphingans have been utilized
for a wide range of biotechnological applications in the food, oilfield, and pharmaceutical industries [4–7]. In recent years, with the continuous exploration of
microbial resources, some new sphingan-secreting strains have been isolated from
diverse environments [8]. Sphingomonas sanxanigenens strain NX02 is a new
species of the genus Sphingomonas sensu stricto that was isolated from soil [9].

Strain NX02 synthesizes a novel sphingan called sphingan Ss, with a linear tetrasaccharide repeat unit consisting of glucose, glucuronic acid, rhamnose, and
mannose [10]. Although sphingan Ss has been used in the field of oil exploitation,
its mechanism of synthesis is still unknown.
The complete biosynthetic pathway of gellan, S-88, and diutan are presently
known. It is a multistep process that can be divided into three sequential steps:
intracellular synthesis of the nucleotide-sugar precursors, assembly of the tetrasaccharide repeat units linked to the inner membrane, and translocation of the repeat
units to the periplasmic space followed by their polymerization and export through
the outer membrane [11–13]. Polymerase, encoded by the spsC gene, catalyzes the
tetrasaccharide repeat units to polysaccharide. The spsC protein involves in
sphingan polysaccharide chain length determination [14, 15].
In this paper, a mini-Tn5 transposon mutant strain of S. sanxanigenens NX02,
which cannot produce sphingan Ss, was screened and isolated. The complete ORF
sequence of spsC gene was obtained by TAIL PCR. The phylogenetic relation and
protein characteristic was analyzed with bioinformatics method.

1.2 Materials and Methods
1.2.1 Bacterial Strains, Plasmids, and Growth Conditions
Escherichia coli strains DH5a (Transgen, Beijing, China) were used as host cells
for gene cloning. E. coli strains S17-1(mini-Tn5) were used as donor strains for
transposon mutagenesis. S. sanxanigenens strain NX02 was cultured on NK
medium (15 g glucose l−1, 5 g peptone l−1, 3 g beef powder l−1, 1 g yeast extract
l−1, and 15 g agar l−1, pH 7.0) at 30 °C. The fermentation medium contained the
following: 45 g glucose l−1, 2.5 g NaNO3 l−1, 0.2 g yeast extract l−1, 1.2 g K2HPO4
l−1, 1 g CaCO3 l−1, 0.005 g FeSO4 l−1, 0.4 g NaCl l−1, and 1 g MgSO4 l−1, pH 7.5.
pEASY-Blunt (Transgen) was employed as gene cloning. When required, the
culture medium was supplemented with 100 mg ampicillin l−1, 30 mg chloramphenicol l−1, or 10 mg kanamycin l−1. Peptone, beef powder, yeast extract, agar,
and other chemicals were purchased from Dingguo Limited (Tianjin, China).


1 Cloning and Bioinformatics Analysis ...

5

1.2.2 Transposon Mutagenesis
Suicide plasmid with transposon mini-Tn5 was transferred from donor strain E. coli
S17-1 into recipient strain S. sanxanigenens NX02 by mobilization with a filter
mating technique [16]. E. coli S17-1(mini-Tn5) was incubated for 12 h at 37 °C with
10 mg kanamycin l−1, and S. sanxanigenens NX02 was incubated for 24 h at 30 °C
with 30 mg chloramphenicol l−1. Filters with the mixture of donor and recipient
strains in a 1:4 ratio were incubated for 8 h at 30 °C on the surface of NK medium
plates. Cells were then suspended in 10 mM MgSO4, and the appropriate dilutions
were plated on selective medium with kanamycin and chloramphenicol. The miniTn5 transposon mutant strains were screened by the viscous phenotype of colony.

1.2.3 DNA Techniques
Standard procedures, including DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, DNA ligation, transformation of E. coli, and S. sanxanigenens, were performed using conventional methods [17]. Genomic DNA was
extracted by LiCl precipitation [18]. Plasmid DNA was purified from E. coli by the
alkaline lysis procedure or using the AxyprepTM Plasmid Miniprep Kit [19].

1.2.4 Cloning and Sequence Determination of spsC Gene
The flanking sequences of mini-Tn5 transposon insertion site was obtained by
the method of Tn5 external direction PCR amplification and long primer RAPD,
using the following primers: Wt1 (5'- CAATAGCGTTATCAACCCGCT-3'), Wt2
(5'-CCAAACGTTGACACCCAGTT-3'), Ric1(5'-ATGTAAGCTCCTGGGGATTCAC-3'), Ric2(5'-AAGTAAGTGACTGGGGTGAGCG-3'), Box(5'-CTACGGCAAGGCGACGCTGACG-3'), Rep1(5'-IIIICGICGICATCIGGC-3'), Rep2 (5'-ICGIC
TTATCIGGCCTAC-3'). The PCR product was sequenced, and analysis of the
deduced amino acid sequence confirmed that it contained an incomplete open
reading frame (ORF) and that the deduced amino acid sequence was homologous to
GelC protein sequences in data banks. The complete ORF sequence of spsC was
obtained by thermal asymmetric interlaced (TAIL) PCR.

1.2.5 Sequence Alignment and Bioinformatics Analysis

Sequence similarity searches were performed using BLAST 2.0 [20] at NCBI.
Alignments to determine protein and DNA similarities were performed using the
CLUSTAL method [21] and a phylogenetic tree was constructed using MEGA 4.0


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X. Li et al.

[36] with the neighbor-joining method [22]. Sequence data were analyzed with
DNAMAN 5.0 (Lynnon Biosoft, Quebec, Canada). The physicochemical and
hydrophobic properties of protein spsC were obtained with program ProtParam and
ProtScale, respectively. The protein secondary structure prediction was analyzed
with program PSIPRED [23].

1.3 Results and Discussion
1.3.1 Tn5-Induced Sphingan Ss-Deficient Mutants
of S. sanxanigenens NX02
A library of random mini-Tn5 insertions was constructed in S. sanxanigenens
NX02 as described in the experimental section. Colonies were individually
screened for sphingan Ss deficient at NK medium plate with chloramphenicol and
kanamycin. The morphological character of wide-type strain NX02 was convex and
viscous (Fig. 1.1a). The flat and tenuous colony of mutant T163 indicated that
sphingans Ss was not secreted from the mutant (Fig. 1.1b). This result was then
confirmed by shake flask fermentation experiment. The result of PCR showed that
the phenotypic change of mutant T163 was caused by mini-Tn5 insertion.

1.3.2 Cloning of spsC Complete ORF Sequence
The flanking sequences of mini-Tn5 insertion site were amplified by PCR. As
shown in Fig. 1.2, two electrophoretic bands of about 1,200 and 1,100 bp were

obtained (lane 1–2). With DNA sequencing and TAIL PCR, the complete ORF

Fig. 1.1 Colony characteristics of strain NX02 and transposon mutant T163


1 Cloning and Bioinformatics Analysis ...

7

Fig. 1.2 PCR result of spsC
gene

sequence of spsC was obtained as shown in lane 3. The nucleotide sequence of
spsC gene has been deposited in the GenBank database under the accession number
AGQ04616.
Nucleotide sequencing of spsC in S. sanxanigenens revealed a unique 1,482-nt
ORF, starting with a putative ATG start codon. Preceding the start codon (8 nt
upstream), a putative ribosome-binding site (RBS) (5'-GGGGA-3') was identified
by taking into consideration previous descriptions of RBSs from S. paucimobilis
ATCC31461 [14]. However, typical −10 and −35 regions were not identified
upstream of the predicted Shine-Dalgarno (SD) sequence. The spsC gene has a high
G +C content (68 %) and a high frequency of G or C in the third position (87 %),
which is characteristic of Sphingomonas genes [24] and consistent with that of S.
sanxanigenens [25].

1.3.3 Phylogenetic Analysis of spsC Amino Acid Sequence
The putative amino acid sequence encoded by the spsC was compared with data
deposited in the GenBank database. The following high levels of identity with other
proteins from a variety of organisms were detected: 60 % identity with polysaccharide biosynthesis protein of Novosphingobium lindaniclasticum LE124
(EQB15321) and 58 % identity with Sphingomonas sp. LH128 (EJU14361), followed by 55 % identity with Novosphingobium sp. AP12 (EJL23329), 52 and 51 %

identity with protein from Sphingomonas sp. PR090111-T3T-6A (WP_019832308)
and Sphingobium sp. YL23 (WP_022681617). Construction of a phylogenetic tree
for the spsC proteins (Fig. 1.3) revealed two obviously divergent phylogenetic
groups of prokaryotes. spsC of S. sanxanigenens was in the group including protein
of N. lindaniclasticumand LE124 and Sphingomonas sp. LH128, but further apart
from the group including protein of Sphingopyxis baekryungensis and Sphingomonas wittichii RW1. Homologous analysis showed that the most sequence of spsC


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X. Li et al.
100
100
99
100
50
100
96
100

Sphingomonas sp. LH128(EJU14361)
Novosphingobium sp. AP12(EJL23329)
Novosphingobium lindaniclasticum LE124(EQB15321)
Sphingomonas sanxanigenens NX02 (AGQ04616)
Sphingobium sp . YL23(WP_022681617)
Sphingomonas sp. PR090111-T3T-6A (WP_019832308)
Sphingopyxis baekryungensis (WP_022672563)
Sphingomonas wittichii RW1(ABQ68766)
Sphingomonas sp. KC8(WP_010125121)
Sphingomonas sp. MM-1(AGH48397)

Syntrophus aciditrophicus (ABC76314)

0.1

Fig. 1.3 Phylognetic tree of spsC amino acid sequence

had high identity with GumC protein which involved in exopolysaccharide Xanthan
biosynthesis, and the N-terminal sequence of spsC had high identity to chain length
determinant protein of Wzz superfamily.

1.3.4 Properties of Protein spsC from S. sanxanigenens
The protein spsC gene is composed of 493 amino acids, with a calculated molecular
mass of 53.51 kDa and a predicted isoelectric point (PI) of 9.42. Analysis of the
amino acid composition of spsC revealed a composition of 54 % polar residues and
46 % hydrophobic residues. The amount of basic and acidic residues was 66 and
55. The result of hydrophobic analysis showed that the aliphatic index of spsC was
0.93, and the instability index was computed to be 52.49 (Fig. 1.4). The analysis of
PSIPRED showed that spsC have two transmembrane domains flanking a central
4
3

Score

2
1
0
-1
-2
-3

1

36

71

106

141

176

211

246

281

Position

Fig. 1.4 Hydrophobicity and hydrophilicity analysis of spsC

316

351

386

421

456


1 Cloning and Bioinformatics Analysis ...

9

extracellular segment. The determination of length distribution of the polysaccharide chains is controlled by a family of proteins termed polysaccharide polymerases
(PCP). PCP enzymes involved in extracellular polysaccharides synthesis systems in
Gram-negative bacteria have, in addition to the membrane/periplasmic domain, a
cytoplasmic domain of protein tyrosine kinases, and the prototype of this family is
Wzc from Escherichia coli [26]. The PCP enzyme in S. sanxanigenens NX02 is
encoded by the gene spsC. The hydrophobic plot for spsC suggested the presence of
two putative transmembrane α-helices, located between amino acids Y13-V44
and P411-L436, respectively. The protein of spsC shows the typical PCP N- and
C-terminal transmembrane helices separated by a segment with a predicted coil
region located in the periplasm.

1.4 Conclusion
Sphingomonas sanxanigenens NX02 is a new species of the genus Sphingomonas
and has low homology with other known sphingan producing strains. The complete
ORF sequence of sphingan gene cannot be obtained by standard PCR with
degenerate primers. Screening deficient mutants is the necessary way to obtain the
gene information about sphingan Ss. In this study, the complete ORF sequence of
spsC gene from S. sanxanigenens was cloned and characterized for the first time.
Bioinformatics analysis showed that the sequence of spsC had high identity with
GumC protein and chain length determinant protein of Wzz superfamily. The
protein of spsC showed the typical polysaccharide polymerases family transmembrane helices and periplasm coil region. This work should prove useful for further
research into sphingan Ss synthesis pathways and genetic engineering with a view
to control sphingan Ss production.

Acknowledgments The authors are grateful to Tianjin Research Program of Application Foundation and Advanced Technology (11JCZDJC16600) for the financial support for this work.

References
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Chapter 2

Preliminary Study on Salt Resistance
Seedling Trait in Maize by SRAP
Molecular Markers
Chunyang Xiang, Jin Du, Peipei Zhang, Gaoyi Cao and Dan Wang

Abstract In this study, different genotypes of maize salt tolerance inbred line and
salt sensitive inbred line were used as the parent hybrid combinations to obtain F2
populations. Two salt tolerance extreme types of DNA pools were established,
where BSA method was used to select polymorphic SRAP markers. The result
showed that 48 pair primers can be amplified and clear and stable bands can be
obtained by parental, tolerant, and sensitive gene pools. Six pair primers of M2E1,
M2E7, M6E15, M7E7, M11E4, and M14E6 showed polymorphism between two
parents and between tolerant and sensitive bulks. The six SRAP molecular markers
closely linked to salt tolerance were determined. The best maize SRAP-PCR

reaction system was established. This research will accelerate maize markerassisted selection breeding and lay the foundation for salt-tolerant gene cloning.
Keywords Maize


Salt tolerance
SRAP molecular marker

In China, salt-affected soils are found in large areas [1]. Saline soil is one type of
middle and low yield soil. It not only affects the growth of plants, but also limits the
yield and quality of crops [2]. Improving and reusing salinized soil have played an
important part in increasing agricultural production. Maize is an important food and
feed crop in China, however, the salt tolerance of maize is relatively poor. Some
researchers have proposed extreme salinity at 0.017 mol/L NaCl [3]. Therefore,
cultivation of resistant maize salt has been attracting considerable attention.
Sequence-related amplified polymorphism (SRAP) is a kind of newly developed
molecular marker system with advantages of stabilization, simplicity, high codominance, moderate throughput ratio, and easily obtainable sequence of selected
bands. Especially, it can be amplified by PCR without any sequence information
[4]. SRAP markers were mainly studied in domestic maize that revealed the genetic
diversity and heterotic grouping of maize germplasm using SRAP markers [5].
Although studies on SRAP markers of salt tolerance in maize have been few for a
C. Xiang (&)
J. Du
P. Zhang
G. Cao
D. Wang
College of Agriculture and Resource and Environment, Tianjin Agricultural University,
Tianjin 300384, China
e-mail:
© Springer-Verlag Berlin Heidelberg 2015
T.-C. Zhang and M. Nakajima (eds.), Advances in Applied Biotechnology,
Lecture Notes in Electrical Engineering 332, DOI 10.1007/978-3-662-45657-6_2

11


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C. Xiang et al.

long time, some researchers have studied it in other plants. Seven SRAP molecular
markers closely linked to salt tolerance were obtained in zoysiagrasses [6]. Its
research can not only provide theoretical guidance for breeding of salt tolerant
maize, but also benefit the selection and cultivation of new varieties of salt-tolerant
maize. Salt resistance seedling traits of maize that were preliminarily studied by
SRAP molecular markers in this research provide the scientific basis for gene
mapping studies of salt tolerance in maize.

2.1 Materials and Methods
2.1.1 Experimental Materials
The F2 segregated population originating from the selfing of F1 hybrids, and F1
from a cross between maize inbred line N1 (A060233, salt tolerant) and M2
(A06148, salt sensitive).

2.1.2 Experimental Methods
2.1.2.1 DNA Extraction and DNA Pools Construction
Total DNA in F2 generations seedlings of maize was extracted with improved
CTAB.
The two relative DNA pools (salt-tolerant DNA pool and susceptible DNA pool)
which come from the F2 populations were made according to the method of BSA
(bulked segregant analysis).

2.1.2.2 SRAP Analysis
SRAP Primer
SRAP primer was designed with reference to the literature [4, 7, 8], and synthesized
by Sangon biotech Shanghai Co. Ltd. as shown in Table 2.1.

PCR Amplification System
The total volume of the reaction system was 20 μL, and concentration of the
components was according to Table 2.2.