Nghiên cứu khả năng sinh tổng hợp IAA của chủng RH

NGHIEN
CQU
KHA NANG
SINH
TONG HOP
lAA
CUA CHUNG
RH
NGUYEN
THI
KIM QUY,
NGUYEN
THI HOAI
HA, PHAM
VAN
TY
Trung
tarn
Cong nghe Sinh hge, DHQG, Ha Ngi
1.
Md
dau
Vice
sir
dung phan bdn vi sinh se lam tang giau dit mot cich ed
hieu qua va do ton kem ban vice su dung
cac loai
phan hda hge, nhiing
loai
cd the gay hai cho mdi trudng vi
lim

can kiet cic ngudn nang
lugng
khdng the tii tao. Nhieu nghien
eiiu
da tap trung cbgn
Igc
cic vi
sinh vat cd
ich
ggi la "vi sinh vat hiru hieu", gdm bdn hgp vi sinh vat,
sdng
edng
sinh vdi nhau nhu trong tu nhien, bao gdm cic vi sinh vat
ehuyen hda photphat khd tan (ehuyen hda
thiie
an kbd tieu thanh
dh
tieu),
vi khuan phan giii chit him
co,
vi khuan lactic sinh bacterioxin
khing vi sinh vat gay benh vi cic vi khuin cd khi nang sinh chat
kich
thich sinh trudng
thue
vat.
Chiing
bd trg nhau phit trien vi phit buy
tic dung nhieu mat
phue

vu cho vice
thiic
diy sinh trudng ciia cay,
xir
ly
miii hdi. Cic vi sinh vat cd trong phan bdn sinh hge con tiet mot sd
chit
kich thieh
sinh trudng, chit hoi hge ehdng nam, ciing nhu lam
tang su nay mim eua hat vi su sinh trudng ciia
xh.
Vice
sir
dung phan
bdn vi sinh se lam tang giau dit mot cich ed hieu qui va dd ton kem
ban
viee sir
dung cic loai phan hoi hge,
nhflng
loai cd the
gay
hai cho
mdi trudng va lim can kiet cic ngudn nang lugng khdng the tii tag.
Vi vay viec nghien
ciiu,
tuyen cbgn vi sinh vat cd boat tinh cao,
vdi cic
ehiie
nang
khae

nhau cung ly
lieh
eu the de lam phan bdn cho
cay trdng la rit cin tbiet, nhim xay dung mot nen ndng nghiep sach,
an toan va
bin
virng. Day la
phuong
hudng rit quan trgng de giim
168
bdt tic hai ciia cic chit hoi hoc giim thieu d
nhidm
mdi trudng va
giam gia thanh sin phim do phai chi
phi
qua nhieu ngoai te nhap
khiu
thud'c
hoi
hge.
Vdi mong mudn gdp phin trong edng nghe san xuit
che
phim phan
htm ea vi sinh da
cbiic
nang va hoin tbien ban,
cbiing
tdi da tien hinb
dt
tii

"Nghien eifu
khd ndng sinh tong hgp
lAA
eua ehung Rh".
2. Nguyen lieu va
phi/dng
phap
2.1. VI sinh vat
Chiing vi khuan phan lap tir dit dugc
giiJ
vi bio quin tai Bao ting
Gidng
chuan
Vi sinh vat, Trung tam Cdng nghe Sinh
hge
- Dai
hge
Qudc gia Hi Ngi.
2.2. Trinh
ti/cac
doan mdi
dCing
trong nghien cuu
27f 5' - agagtttgatcctggctcag - 3'
780f
5'
- GATTAGATACCCTGGTAG -
3'
920r
5'-GTCaaTrCCTTTGAGTTT-3'

1525r 5' - aaaggaggtgatccagec - 3'
10
X
Taq buffer dNTP mix 2.5mM
Taq polymeraza (5U).
2.3. M6I
trwdng
(YEM,
g/l)
[64]
K2HPO4-l,0,
MgS047
H2O-O,
4,
NaCl-0,2,
Mannitol-20,0.
Kbit
trung d 12rC/15phut, pH
7;2.
2.4. Phuong phap xac dinh
lAA
(indole - 3 - acetic - acid)
[44,45,62]
3. Ket qua va thao
luan
3.1. Tuyen chon ehung
Rhizoblum sinh lAA
sau 42
gid nuoi
bang

phuong phap sac ky ban mong
Thieu hut boocmon sinh trudng
li
van de ma
cay
trdng
thudng
gap
phii.
Dieu niy cd the khic phue dugc neu bd sung vao dit
viing
rd
cic chiing vi khuan cd boat
tinh
sinh cac chit kich
thich
sinh
trudng cao.
169
c
•a,
<
<
B
X
26
2
il
I
16

24
21 III
21
18
17
OHmd lUffng lAA (m|/1)
Ky hieu chiing
Hinh
1.
Tuyen chon cac chiing vi khuan c6 kha nang sinh
lAA
Nhieu nghien
eiiu
da tim hieu kha nang cd dinh nita ciia vi khuan
vung
r6
Rhizobium [4, 5]. Trong nghien
eiiu
niy chiing tdi quan tam
den kha nang hinb thanh
lAA
ciia chiing. Tir cic miu dit trdng ngd
vi dau xung quanh Ha Ngi ehung tdi tuyen cbgn dugc 10 ehung cd
khi nang sinh tdng hgp
lAA
cao. Axit indon axetie
(lAA)
li mot
chit kich thich sinh trudng thue vat. Hoocmon dugc mot vii vi sinh
vat sin sinh ngay tai

viing
tiep
xiic
vdi be mat
Idng
hiit,
do dd, cay
trdng cd the su dung mot cich cd hieu qui.
Trude
day ngudi ta cho
ring khi nang kich thich sinh trudng ciia nhdm vi khuin niy la do
qui trinh cd dinh nita.
Nbung sau dd bing thue nghiem ngudi ta da cbiing minb ring ngay
trong nhiing dieu kien mi qui trinh cd dinh nita khdng
di6n
ra vin xiy
ra su
kich
thich sinh trudng thue vat. Ket qui cho thiy ea 10 ehung
(Hinh 1) deu cd kha nang sinh
lAA
rd ret. Tuy nhien, him lugng
lAA
ciia cic chiing vi khuan sinh ra la khdng gidng nhau
13-27mg/l.
Dae biet cd cbiing ky hieu Rh ed khi nang tdng hgp
lAA
him
lugng dat
tir

27mg/l.
Viec tuyen cbgn cac chiing vi khuan chi la khau
diu tien trong cic bude tuyen cbgn cic dac tinh ciia vi khuan. Dich vi
khuan dugc nudi 56 gid d nbiet do
30°C.
1ml dich miu dugc ly tam
lanh 4°C
vdi tde do 10000vdng/15phut. Loai bd dich trong.
170
Hoa tan can vdi
100|il
metanol 100%.
Diing
mao quan chim toan
bd dieb miu
Idn
sic ky ban mong va
5|il
eiia lAA
(Sigma) dugc dung
lam thang chuin [45, 46].
Viec phan
tich lAA
bang phep sic
ky ban mong muc dich chiing tdi dung
de tich
chinb
xic lugng
lAA
trong dich

nudi ciia chiing Rh vi (Hinh 2) cbiing
bieu hien khi rd bang boat tinh khi
nang sinh
lAA
tuang
ling
vdi chim
lAA
chuin. Tien hinh cao vet d vi tri
sd 3 li ciia chiing Rh.
Cbiet miu va tinh sach miu bing
^*''*
lOOfil
metanol trong dng nghiem cd
niit 1234
xoay. Lge qua ban
Igc
0,45|am.
Ly tam
Hinh
2.
Xac dinh
lAA ciia
chiing
lanh
4"C
d 10
OOOvdng/phiit
trong Rh bang sac ky ban mong
20phut.

Bd can,
liy
dich ndi, cho bay hoi tdi khi miu kbd. Bd sung
lai 20)0,1
metanol vio miu vi giir d
4"C.
Sau dd, miu dugc dung cho viec xac dinh
him lugng
lAA
cd trong miu tren sic ky long cao ap (HPLC).
Viec
thu
phan doan
lAA
qua sic
ky long cao ap (Hinh 3)
ciia
chiing
li khdng gidng nhau ve tbdi gian,
chi sau 10
pbiit
da xuit hien dinh
lAA
chiing Rh.
Cic phan doan thu dugc tien
hinb dinh lugng
lAA
tren miy quang
pbd. Dinh lugng dich thu dugc d 2
dng nghiem, cho vao dng

1:1
ml
lAA,
dng II (dd'i
chiing):
1
ml
nude
cat. Them vao mdi dng 2ml thud'c
thit
Van Urk Salkowski (0.5M
FeCl^
1ml, 35%
HCIO4
50ml) phan irng
dugc
lie
deu. Dung dich cd miu
hong nhat sau 15-30
pbiit.
Thdi gian
(phiil)
Hinh 3. Hinh hap phu
lAA ciia
chiing Rh tren bieu d6 HPLC.
171
Chi sd OD dugc dd'i
chi6'u
vdi phuong trinh dd thi chuin de
tinh

ham lugng
lAA
cd trong dich nudi. Dieu chiing tdi nhan thiy d day rit
rd li cbiing Rh li 40mg/l. Dieu niy phii hgp vdi nhieu nghien
eiiu
trong va ngoii nude ve kha nang sinh
lAA
eua cic chiing vi khuan
viingri
[6].
3.2. Dac diem hinh
thai,
sinh ly, sinh hoa
eua ehung
Rh
Nhu chiing ta biet, the gidi vi sinh vat bao gdm mot sd lugng
Idn
cic
loii
khic nhau, chiing khdng the dugc gdp ehung lai vao mot
nhdm phan loai nao dd. Tit ea cic dd'i tugng nay deu li cic ea
th6'
hien vi, phan
Idn
li don bio, cbiing dugc nudi eiy va nghien
cihi
theo
nbttng
phuong phap tuang tu nhau.
Chiing rit da dang ve hinh thii hge, ngay ea khi cac loii gidng

nhau ve hinh thii ciing cd the khic nhau rit rd ret ve cic dac diem
sinh ly, sinh boa hge. Vi vay, nhieu vi sinh vat dugc xep vao cac nhdm
sinh ly khic nhau khdng phu
'thude
vio vi tri phan loai ma phu
thude
vao
tac
dgng ciia chiing
len
mot
-cbit
nao dd ciia ea chit hoae vao ban
chit eua cic sin pham trao ddi chit dugc tao ra. Do vay, 2 ehung HA4
va Rh cung khdng nim ra khdi qui
luat
ehung dd. Hai chiing niy dugc
nudi tren cic mdi trudng dac trung cho tirng loii nhu da md ti d phin
phuong phip.
- Chiing Rh cd hinh que kich thude
0,5-0,9x1,20-2,0|j,m,
pbd bien
la da hinh trong nhirng dieu kien bit
Igi.
Thudng
ehiia
nbung hat
poly-|3-hydroxybutyrat,
nhiing hat nay
khtic

xa tia sang khi quan sat dudi kinh hien vi phan pha. Vi khuin
Gram (-), hieu khi, cd khi nang di dgng. Khuan lac hinb trdn,
Idi,
nhiy, thudng cd dudng kinh 2-4mm. Cd kha nang ddng boa ngudn
eacbonhydrat nhu: Mantoza, saccaroza, glucoza, fructoza, xyloza,
raffinoza,
galaetoza, khdng sinh trudng dugc d ndng do
NaCl
2%,
khdng sinh
HjS.
Mot dieu nhan thiy d day la ea 2 ehung deu ed the
tdn tai dang cyst khi gap dieu kien bat
Igi
xiy ra, ehung thude vi
khuan Gram am.
Khuan lac eua chiing deu nhiy vi khd lay khi tich. Han niia.
cbiing Rh deu cd khi nang ddng hoi cic loai dudng pbd bien dung
cho cic vi khuan nhu: Mantoza, saccaroza, glucoza, fructoza
172
Hinh
4.
Hinh thai khuin
lac ciia ehung
Rh
Hinh 5.
Hinh
dang te bao
ciia
chiing Rh

(X7.500)
Can
cii
vio cic dac diem hinh thii (Hinh
),
sinh ly, sinh hoi, ddng
dua vao
khoi
phan loai Bergeys [66], ehung tdi xic dinh cd kha nang
cbiing Rh thude chi Sinorhizobium.
De khing dinh
cbinb
xac den loai ehung Rh
thude
loii nao trong
chi Sinorhizobium chiing tdi tien hinb tich cbiet ADN
ciia
cbiing Rh
ddng thdi tien hinb phin
ling
PCR.
1
M
"2"
1500 -2000 bp
Ket
time
qud trinh phdn
ling
sau

30 chu ky
khue'eh
dqi, ehung Rh tong
hgp dugc doqn gen ed kich thude nam
trong khodng
tH
1500 - 2000bp (Hinh
6).
San pham ndy dugc dung lam
khuon cho viec phdn tich trinh tu gidi
trinh turADN
16S.
Hinh 6. Dien di dd tren gel agaro
10% sdn pham PCR
ciia
ehung Rh
Chii
thieh: M ADN chuin
Ikb
Ladder
2 - Chung Rh
Tien hinh phan tich trinh tu
rADN
16S eua Rh tren may dgc trinh
tu tu dgng
3100 Avant
bing doan mdi
dac
bieu
ciia

16S. Chiing tdi
173
dung cac cap mdi thdng dung va pbd bi6'n ma cac phdng
thi
nghiem
trfin
the gidi vin dang dung, bao gdm cic doan 27F, 780F, 980R va
1525R.
Diing
ehuang trinh Blast search ket qui phan tich trinh tu cho
thiy doan gen dugc tdng hgp eua Rh ed do dii li 493bp day la
nhtrng
doan gen bao tdn va cbgn lge trong qui trinh
diing
4 mdi cho phan
tich trinh tu song song. Khi so sinh Rh vdi trinh tu gen ma hoi cho
16S da dugc dang ky trong ngan hing dir lieu gen Nhat Ban. Do tuang
ddng rit cao dat 97% vdi trinh tu
ciia
ehung thude loai Sinorhizobium
xinjiangensis vdi ky hieu AJ420774, day la nhirng chi so tuang ddng
so sanh dugc phep chip nhan. Tit cac thdng sd thu dugc eua
Sinorhizobium xinjiangensis Rh se lam co sd va cin tbiet cho viec lap
so dd
cay
phi he (Hinh 7).
Mdi quan he gin giira chiing
xinjiangensis
Sinorhizobium xinjiangensis Rh
vdi 14 trat tu nucleotit thude

Rhizobium trong ngan hang dir
lieu gen Nhat Ban. Sir dung
ehuang trinh dien toan MEGA 2.1
va cac thdng sd tien hoi cin tbiet
cho thiy mdi quan he da dugc
tbiet lap tren ea sd phan tich so
sinh chudi trinh tu nucleotit doan
gen rADN 16S dii493bp. Cho
thiy ehung Rh nim tren cung mot
nhinh va cimg nhdm vdi loai
Sinorhizobium xinjiangensis ky
hieu AJ420774.
Trong cic budc nghien
eiiu
thi viec
lua
cbgn cac dieu kien
nudi ciy va cac yeu td inh hudng
den qua trinh sinh trudng eua cic
ehung vi khuan phan lap dugc li
mot viec rit quan trgng vi khdng
the thieu.
S.melilotiAYI
96963
S.frediiAY260149
S.frediiAY260150
S.melilotlD14509
R.melilotiX67222
R.fredilX67231
S.sahellX68390

Rh
S.xlnjlangenslsAF250354
Rh.melilotiD12783
Rh.fredilD12792
S.fredliD14516
- S.kummerowlaeAF364067
S.morelenseA Y559079
S.morelenseA Y024335
H
Hinh 7. Cay pha he dua tren phan tich
trinh
tu rADN 168 ciia chiing Rh va cac
loai
CO
quan he
gdn
174
Vdi muc dich nghien
eiiu
phdi trdn bdn hgp cic cbimg vi khuan
trong che pham phan
hiJu
ca da
chiie
nang, thi khdng the khdng nghien
ciiu
ky dac tinh
ciia
tirng cbiing dugc.
Do vay,

ehiing
tdi ciing tien hinh nghien
ciiu
tiing chiing cd khi
ket hgp phan tich cic chiing vdi nhau mong mudn tim ra dugc cac dieu
kien thieh hgp cho sinh trudng va tdng hgp
lAA ciia
chiing Rh.
Ket luan
Tir cic miu dit trdng dau da phan lap dugc cbiing vi khuin Rh cd
khi nang kich thich sinh trudng thue vat. Dua vao phuong phip phan
loai truyen thdng theo khoi phan loai eua Bergey's va phuang phip
phan loai hien dai dua tren phan tich trinh tu rADN 16S, ehung vi
khuan Rh thude loai Sinorhizobium xinjiangensis. Chiing Rh cd kha
nang sinh chat kich thich sinh trudng thue vat dat 45mg/l.
Summary
Study on Indole
3-Acetic
acid production of bacteria Rh
Nguyen Thi Hoai Ha, Nguyen Thi Kim Quy, Pham Van Ty
Center of Biotechnology, Vietnam National of University Hanoi
The strain of Rh was isolated from soil cell are rods, non sporing,
non-notile,
catalase negative. Metabolision was predominantly
fermentative, lactic acid being formed from glucose homofermentatively,
xylose was fermented. The analysis of 16S rDNA sequence shows that
this strain Rh was identified as Sinorhizobium xinjiangensis.
lAA
was
produced.

Tai lieu tham khao
1.
Hoben H. J., Somasegaran P., Handbook for
Rliizobia,
Springer-
Verlag, New York, pp. 1-6.
2.
Holt J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T.
Williams (1994), Bergey's Manual of Systematic Bacteriology,
Williams and Wilkins. Baltimore, Maryland, pp. 235-242.
3.
Lynch, J. M. (1990), "Some consequences of microbial
175
Rhizosphere competence for plant and soil". In The
Rhizosphere, ed. J. M. Lynch, pp. 1-10.
4.
Michalczuk L., Ribnicky D. M., Cooke T. J. and Cohen J. D
(1992),
"Regulation of indole-3-aeetic acid biosynthesis in
carrot cell
cultures".
Plant Physiol, (100), pp. 1346-1353.
5.
Ribnicky D. M., Cohen J. D., Hu W. S. and Cooke T. J. (2002),
"An auxin surge following fertilization in carrot: Its
significance for plant totipotency", Planta, (214), pp. 505-509.
6. Ridge R. W. and B. G.
Rolfe
(1985),
''Rhizobium

sp.
degradation of legume root hair cell wall at the site of infection
thread origin",
Appl.
Environ. Microbiol, 50, pp.
717-720.
7.
Vincent, J. M. (1970), A Manual for the Practical study of the
Root-Nodule
Bacteria,
Burgess and Son Ltd. Great Britain, pp. 45.
8. Visser E. J. W., Cohen J. D., Barendse G. W. M.,
Blom
C. W.
P.
M., Voesenek L. A. C. J. (1996), "An ethylene-mediated
increase of sensitivity to auxin induces adventitious root
formation in flooded Rumex
palustrisSm.
Plants", Plant
Physiol,
(112),
pp. 1687-1692.
176