PHD thesis abstrac isolation, point mutation of P5CS gene conferring drought tolerance and transformion into soybean varieties of vietnam

MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY NGUYEN THI THUY HUONG ISOLATION, POINT MUTATION OF P5CS GENE CONFERRING DROUGHT TOLERANCE AND TRANSFORMION INTO SOYBEAN VARIET

MINISTRY OF EDUCATION AND TRAINING

THAI NGUYEN UNIVERSITY

NGUYEN THI THUY HUONG

ISOLATION, POINT MUTATION OF P5CS GENE CONFERRING DROUGHT TOLERANCE AND TRANSFORMION INTO SOYBEAN VARIETIES OF

VIETNAM

Major: Genetics Code: 62.42.70.01

PHD THESIS ABSTRACT

THÁI NGUYÊN - 2011

PUBLICATIONS

1 Nguyen Thi Thuy Huong , Chu Hoang Mau, Ha Tan Thu, Dinh Thi Kim Phuong,

Tran Thi Truong (2006), “Collection, classification, and qualitative assessment

of some local soybean cultivars in Sonla province, Science & Technology

Journal of Agriculture & Rural development, 11 (1): 28-32

2 Chu Hoang Mau, Nguyen Thi Thuy Huong (2006), “Amino acid content and

drought tolerance of some local soybean variables in Sonla province ”, Science

& Technology Journal of Agriculture & Rural development, 20 (2): 22-26

3 Nguyen Thi Thuy Huong, Chu Hoang Mau, Le Van Son, Nguyen Huu Cuong, Le Tran Binh, Chu Hoang Ha (2008), "Evaluation of drought tolerant ability and

cloning of P5CS gene of some soybean cultivars (Glycine max L.Merrili)",

Journal of Biotechnology, 6(4): 459-466

4 Nguyen Thi Thuy Huong, Tran Thi Ngoc Diep, Nguyen Thu Hien, Chu Hoang Mau,

Le Van Son, Chu Hoang Ha (2009) "The development of in vitro regeneration

system from cotyledonary node for transformation in soybean (Glycine max L.Merrili) ", Journal of Science and Technolog, Thai Nguyen University 52(4):

Bio-Hanoi 2010 Journal of Biotechnology, 8 (3A): 539-544

8 Chu Hoang Mau, Nguyen thi Thuy Huong, Nguyen Tuan Anh, Chu Hoang Lan,

Le van Son, Chu Hoang Ha ( 2010) Characteristic of the gene encoding pyrroline

– 5 – carboxylate synthase (P5CS) in Vietnamese sobean cultivar (Glycine max

L.Merrill) 2010 International Conference on Biology, Environment and Chemistry (ICBEC 2010) IEEE: 319-323

INTRODUCTION

1 Preface

Soybean (Glycin max L Merrill) is one of the most important crops not only in Vietnam but also in other contries in the world The soybean of Vietnam and other world-leading producting coutries in the world is seriously affected by drought annually Soybean has low resistance to drought as well-documented so far in many genotypes In Vietnam, scienctists have succeeded in creating new varieties with improved tolerace to drought through traditional breeding and these varieties are widely cultivated in many areas However, the traditional breeding itself possesses disadvantages such as laboriousness, time spending, requiring enough hybrids and unstability The developments of modern biotechnology can overcome these disadvantages of conventional methods On of the well-known technology is plant

transformation mediated by Agrobacterium tumefaciens successfully applied in

varied plant crops by breeding scientist in the world The research group of Dr Tran Thi Cuc Hoa at the Cuu Long Delta Institute of Rice Research has recently reported a transformation system for soybean and the system is applied to produce novel soybean varieties with resistant to differenct pathogens Despite this fact, the application of plant transformation for developing soybean with modified stress tolerance has not been used domestically

Based on above arguments we performe a work entitled “Isolation, point

mutation of P5CS gene conferring drought tolerance and transformation into

soybean varieties of Vietnam”

3.1 Characterization of biochemical and physiological features of soybean varieties

grown in the North of Vietnam

3.2 Isolating and sequencing gene encoding for 1-pyroline-5 carboxylate synthetase

(P5CS)

3.3 Site-directed mutagenization of P5CS in order to remove the feedback inhibition

by proline

3.4 Isolation of promoter rd29A – a responsive promoter under dehydration conditions – from Arabidopsis thaliana and characterization of promoter rd29A in

transgenic tobacco

3.5 Construction of vector carrying P5CS under the control of promoter rd29A and

transformation of this construct into tobacco

3.6 Characterization and analysis of drought tolerance transgenic tobacco lines under

stress conditions in vitro

3.7 Transformation of the construct carrying P5CS under the control of promoter

rd29A into Vietnamese soybean and analysis of the present of P5CS in transgenic

lines

4 Significant results

4.1 Gene P5CS was successfully isolated and sequenced from two Vietnamese

soybean varieties, DT84 and SL5, respectively, with 2148 nucleotides in length and encoded for a protein with 715 amino acids

4.2 The feedback inhibition by proline of enzyme P5CS was successfully removed using site-directed mutagenesis at the 125th amino acid (Aspartate into Alanine)

4.3 Promoter rd29A was successfully isolated from Arabidopsis thaliana with 1298

nucleotide in length carrying typical element of a responsive-dehydration promoter such as MYB, DRE, AMYBOX

4.4 The vectors carrying GUS and P5CSM under the control of promoter rd29A were

constructed and transformed into tobacco The transgenic tobacco lines containing

construct rd29A::GUS showed significant enhancement in enzyme activity of GUS

under stress conditions Moreover, the transgenic tobacco lines containing construct

rd29A::P5CSM represented significantly higher resistance to drought in comparison with the wildtype plants The results confirmed a fact that this construct can be reasonably used for other crops and plant species in order to create new plant varieties with improved stress tolerance

4.5 The transformation system into soybean was optimized and used to create

transgenic soybean lines containing construct rd29A::P5CSM These lines were

positively analyzed by PCR and will be good materials for further researches on developing new soybean varieties with high resistance to drought

5 Scientific and applied values

5.1 Scientifically, this is the first report in Vietnam on determining the tolerance of varied soybean varieties under artificial stress conditions, isolating and modifying the

structure of P5CS by site directed mutagenesis by PCR The results on analysis of

P5CS confirm the fact that the difference in drought tolerance in soybean is determined by complicated mechanism, however P5CS is one of the most important factors conferring the drought tolerance in crop in general

5.2 Practically, the results on analysis of transgenic soybean plants containing the mutant P5CS under the control of stress inducible promoter rd29 proved the practical values of the thesis

The drought tolerance in transgenic tobacco containing this structure show that this can be applied on other crops in order to improve one the most vital trait of crop, drought The results on establishing the transformation system in soybean are the valued basis for further developing novel soybean varieties with high stress tolerance

6 Structure of the thesis

The thesis includes 113 pages, divided by parts as following: Introduction composes

3 pages; Chapter 1: Overview, 34 pages; Chapter 2: Materials and Methods, 15 pages; Chapter 3: Result and discussion, 46 pages; Outlooks, 2 pages; Publication: 1 page; References: 11 pages; The thesis included 29 tables, 43 figures and 118 references in English and Vietnam

Physiological and biochemical basics of drought tolerance in soybean; (3) Proline and the improtant function of P5CS in the proline biosynthesis in plants; (4) Promoter and roles in controlling the expression of genes under drougth conditions; (5) Studies on improving the drought tolerance of soybean in the world

Proline is known as one of the compounds functioning in osmotic regulation mechanism of plant cell under stress conditions such as drought, salinity (Delauney and Verma, 1993) Beside that, proline plays vital roles in protecting cellular structures and macro molecules when the cell is under osmotic stresses; in protecting protein structure and improving the activity of varied enzymes; in digesting reactive oxygen residues and in inactivating singlet oxygen quencher (Szavados et al, 2009)

In plants, the biotesynthesis of proline is controlled by two genes encoding for P5CS

These two genes are highly homologous in amino acid sequence but expressed differently under different conditions Both genes are found to express in flower in order

to provide proline for the flower development processes (Mattioli et al, 2009) The study

on Arabidopsis showed the tight relationship between the expression of these two genes

and the accumulation of proline when plants are exposed to salinity condition; and the feedback inhibition by poline on P5CS is also affected under the same conditions The

transgenic tobacco overexpressed a mutant P5CS which lost this inhibition showed that

the accumulation of proline increase up to two folds in comparison to wildtype

Recently, a P5CS has been isolated from rice and when transformed back into rice

revealed that transgenic plants showed higher tolerance to salinity and cold

Promoter rd29A is an inducible sequence under varied conditions such as drought, salt and cold Studies found important motifs in rd29A sequence related to the operation such as ABRE and (DRE)/C repeat (CRT) These two motifs are

thought to be important for rd29A in order to control the expression of genes under

stress conditions of surrouding environment (Yamaguchi-Shinozaki và Shinozaki,

1993) Rd29A has been isolated from many plant species such as Arabidopsis,

tobacco and rye (Sun và Chen, 2002) The transformation vectors bearing GUS under

the control of rd29A have been transformed into potato and sugarcane The activity of

GUS has been found in transgenic plants under different stress conditions in culture

(Zhang et al., 2005)

In therory, there are different methods for improving the stress tolerance in plants, however the application of transformation technology is more preferable with breeding scientist For Vietnam, there is no doubt about the high potential of application this technique in developing new crop varities with improve drought tolerance

Chapter 2 MATERIALS AND METHODS 2.1 Materials, chemicals and equipments

Plant materials: 16 local and DT 84 soybean varieties ; 326 tobacco variety

(Nicotiana tabacum), and Arabidopsis thaliana

Chemicals: pBT clone vector, pBI101 vector for contructing a transgenic vector, pTN

289 transgenic vector Primers for amplifying the P5CS gene and the rd29A promoter

were designed based on the nucleotide sequence of P5CS gene (Genebank code:

AY492005) and the nucleotide sequence of the rd29A promoter (Genebank code:

AB428730) The chemicals for tissue culture and bio-molecular experiments belonging to Merk, Bioneer, Fermentas were supported by Plant Cell Biotechnology Department

2.2 Methods

2.2.1 Bio-physical and bio-chemical methods

- The ability of drought tolerance was rapidly estimated following Binh L T et al

1998

- The dissolved protein concentration was determinated by Lowry’s method Amount

of lipids was determinted following Chau P T T Amount and components of amino

acids of seeds were determinted following Chi P V et al (1997)

- Prolin concentration was determinted following Bates et al (1973)

- Data was analyzed following Tuat N H and Khoi N K (1996)

2.2.2 In vitro tissue culture methods

In vitro tissue culture methods on Arabidopsis and tobacco are following

Topping ,1988 Soybean plants were generated by the multiple-shooting system using axillary cotyledon of matured seeds The transgenic protocol using axillary cotyledon of matured seeds was improved from Olhoft’s method (2001)

2.2.3 Bio-molecular methods

- Specific primers were designed based on nucleotide sequences on Genebank

- The total DNA was isolated from arabidopsic and tobacco leaves;

- The total RNA was isolated from soybean using Trizol Regents kit (Invitrogen)

- cDNA was synthesized by using Total RNA and RevertAidTMH Minus First Strand

cDNA Synthesis Kit (Fermentas)

- PCR reaction: P5CS gene was amplified by using specific primers The PCR reaction consisted of 940C for 5 minutes, then 35 cycles of 94°C for 30 seconds, Tm (from

500C to 620C) for 45 seconds, and 72°C for 60 seconds, followed by a final extension

of 72°C for 10 minutes

- OE - PCR reaction( Overlap Extension-PCR)

OE-PCR reaction was carried out following: 94ºC for 5 minutes; then 4 cycles of 94ºC for 30 seconds, Tm (from 500C to 620C) for 45 seconds, 72ºC for 90 seconds; 72ºC for

10 minutes, followed by a final extension of 72°C for 10 minutes Then, the products were placed immediately on ice and 1µl BamHI and 1µl SaclI The OE-PCR reactions were continued following 94ºC for 5 minutes; then 30 cycles of 94ºC for 30 seconds,

Tm (from 500C to 620C) for 45 seconds, 72ºC for 90 seconds; 72ºC for 10 minutes, followed by a final extension of 72°C for 10 minutes

- Cloning methods: recombined plasmids were isolated and purified following Sambrook et al (2001) and Plasmid Miniprep Kit (Qiagen) Cutting by restricted enzymes was done following Sambrook et al (2001)

- Colony-PCR method

- Constructing vectors: rd29A :: GUS và rd29A :: P5CSM

- Transgenic plant analysis methods: the present of inserted gene in transgenic plants was identified by bio-chemical methods Transgenic tobacco plants were treated

drought artificially by using PEG following Jun et al (2001) The concentration of

beta-glucuronidase (GUS) was determinated following Tefferson et al (1987)

Chapter 3 RESULTS AND DISSCUSION 3.1 Results in collection and evaluation of local soybean varieties in Son La province

3.1.1 Characteristics of morphology and biochemistry

Sixteen soybean varieties were detected in seven counties in 11 different districts of Son La province Protein content of these varieties ranged between 29.72% -52.75% protein / dry weight and lipid content ranged from 9.9% - 18.65% in which DT84 varieties have the highest lipid content 18.65%, followed by SL3 (17.34%)

3.1.2 Analysis of drought tolerance ability of soybean varieties

Proline content of SL5 increased up to 377.44% (highest value) after 9 days of drought treatment Whereas proline content of DT84 increased slightly at all three time points (101.06, 129.26 and 146.81%) This result is consistent with the other studies when they research on drought tolerace of different crops such as rice (Nguyen Huu Cuong et al; Due et al; Choudhary et al 2005), legumes (Curtis et al;

Chen et al)

3.2 Cloning of P5CS and elimination of reverse inhibition by site-directed mutation 3.2.1 Cloning of P5CS

3.2.2 Results in amplification, cloning and sequencing of P5CS gene

Total RNA was extracted and cDNA was synthesized from SL5 and DT84 Four nucleotide fragments of P5CS gene were amplified by PCR and checked by electrophoresis on 0.8% agarose gel Figure 3.3 showed that the size of the gain band corresponding to the size of the theoretical calculations In order to obtain full sequence of P5CS gene, these two PCR products were mixed and used as template for PCR using primers P5CSfor/P5CSrev PCR products obtained approximately

2100 bp in size (Figure 3.4)

Figure 3.3 PCR amplification of two

P5CS nucleotide fragments of DT84 and

Sequencing results showed that the nucleotide sequences of both two samples (DT84 and SL5) contain 2148 nucleotides encoding 715 amino acids In which two amino acid Asp125 and Phe128 in the deduced P5CS amino acid sequence cause inhibition

of P5CS activity by increasing proline content in cells (Zhang et al 1995)

2

Figure 3.10 Identification of P5CS by

PCR with P5CS M125for2/SacI-P5C

primer pair (1) and P5CS for / P5CS rev

primer pair (2); M : 1kb DNA ladder

Figure 3.11 Combination of two DNA

fragments following OE-PCR method

using BamHI-P5CS and Sacl-P5CS

primer pair A, B : 1+2, M: 1kb DNA

M A B

M 1 2 3 4

2100bp

M 1 2 3 4