Cloning Of Cotton Gols Gene And The Creation Of New Drought And Salt Tolerant Germplasm

As one of the main economic crops in China,cotton is particularly affected by drought and salinization,which hinders the development of national economy.In recent years,with the rapid rise of genetic engineering,the conventional crossbreeding method has been broken,and the breeding cycle has been shortened significantly,which has become an important means of breeding.ERFs(Ethylene responsive factors),which can be induced to express under abiotic stresses,are important transcription factors in plants.Cottonseed sugar oligosaccharide(Raffinose familyoligosaccharides,RFOs)is a kind of oligosaccharide molecule,which can regulate the osmotic pressure of plants to maintain the osmotic balance in plants.Inositol synthase(Gols)is a key enzyme in the biosyntheticpath of RFOs,which can be induced by drought,high salt and high temperature stresses.In this paper,two transcription factorb genes.Pe DREB and Kc ERF,were isolated from(Populus euphratica Oliv)respectively,The two genes were constructed to a plant expression vector containing Kan resistance.The plant expression vector was used for cotton transformation.In addition,Gh Gols geneswere constructed and connected to the expression vector containing Bar resistance marker gene and transformed into Arabidopsis thaliana for functional verification of GhGols genes..(1)Genes for drought-tolerant responsive transcription factors Kc ERF and Pe DREB2 awere isolated from(Populus euphratica Oliv)respectively.A bivalent plant expression vector harboring Pe DREB and Kc ERF genes was constructed.The construct was used for the transformation of cotton cultivars Guo Xin-3 and Jin-12,using the method of pollen tube channel of transformation.For a a preliminary screening of the transgentic cotton plants,a method of spraying of kanamycin(Kan)solution on the cotton seedling leaves was used.23 T0 generation cotton plants wre selected by this method.The T3 generation cotton plants were further used for tests of gene integration of Kc ERF-Pe DREB genes and drought resistance and salt tolerance of the transgenic cotton plants.(2)the Gols gene was screened by stress,and Ghgols2-2 was selected as apotential gene for further study.Ghgols2-2 was transfered into Arabidopsis thaliana,and 6 strains of transgenic strains were screened.The T3 generation of transgenic A.thalianahomozygous lines were obtained after the propagation.Transgenic plants were treated with 15% PEG and 200 m M Nacl,and their relative electrical conductivity,relative water content,malondialdehyde content and inositol glycosylase activity were measured.After the treatment of 7d by PEG and Nacl,the relative water content of transgenic plants was maintained at 92~93%,while the control group was 85%.The content of malondialdehyde in the control group was 1.5~2.08 times of that of the transgenic plants.Compared with the control group,the relative electrical conductivity of transgenic plants decreased significantly.The activity of inositol and galactosidase activity in transgenic arabidopsis thaliana after 30 d stress was determined,and the activity of inosidase in the transgenic plants was 5.0-6.7 times of the control group.The results showed that the control group was more sensible to abiotic stress than the transgenic plants,i.e.,the resistance of transgenic Arabidopsis was significantly stronger than that of the control group.In addition,after treatment with 15% PEG,and 100 m M,200 m M Nacl,the transgenic A.thalianaplants grow well,while the control groupalmost died.This showed that the transgenic plants with Gh Gols2-2 gene over-expressed A.thalianaplants gained more tolerance to slt and drou Ght.