Screening Of Drought-tolerant Chickpea Germplasm, Construction Of CDNA Libraries, Analysis Of EST Sequences And Clonging Of Water-stress Responsed Genes

With deterioration of global climate and destruction of the balance of biogeocenose, the area of aridity, desertification and salinization rapidly increased. The shortage of water resources has been a serious ecological problem which faced by human being. Drought has also been a global agricultural calamity and is first abiotic stress factor led to reduction of yield.The cultivated chickpea, Cicer arietinum L., was one of the first grain legumes to be domesticated in the ancientry. Today, chickpea is the third most important pulse crop in the world. Due to displaying considerable drought tolerance, adverse resistance and high water used efficiency, chickpea is being used as a important gene resource for improving drought tolerance of crop. In this study, strong drought tolerant varieties were screened and identified from 12 chickpea varieties from Xinjiang Autonomous Region. On the basis of construction of two cDNA libraries of chickpea leaves under water stress and analysis of expressed sequence tags （ESTs）, cloning and expression pattern of some important water-stress responded genes involved in SAM pathway were studied. The main conclusions are as follows:1. Effects of drought stress on physiological-biochemical indexes and identification of drought tolerance of chickpea cultivarsThrough laboratory experiment study, relative water content, malondialdehyde content, proline content and other physiological-biochemical indexes of leaves of 12 chickpea cultivars under drought stress were investigated. The relationship between these physiological-biochemical indexes and drought tolerance were discussed. By system cluster analysis, the 12 chickpea varieties could be classified into 3 drought tolerant ranks:the strong drought tolerant, the medium drought tolerant, and the weak drought tolerant. The results showed that the strong drought tolerant varieties have stronger water retaining capacity, smaller water deficit, larger increase multiple of proline, higher relative integrity of the membrane, smaller membrane lipid peroxidation, smaller decline rate of net photo synthetic, larger decline rate of stomata conductance and transpiration, higher water use efficiency.The IAA, GA3, ZR and ABA contents in chickpea under drought conditions were determined by the method of ELISA and the relationship between these endogenous hormones and drought tolerance were discussed. The results showed, after 60 mM PEG 4000-treated 24 h, contents of ABA and IAA increased significantly and contents of GA3 and ZR decreased significantly in 209 （strong drought tolerant） and 88-1 （weak drought tolerant）. The stronger the drought tolerance of the variety is, the more the decrease of GA3 and ZR contents, the less the increase of ABA and IAA contents.2. Construction and identification of two cDNA libraries of chickpea leavesOn the basis of identification of drought tolerance, two non-normalized cDNA libraries were constructed from the seedling leaves of a strong drought-tolerant chickpea cultivar 209 under PEG 4000-treated （MH1） and-nontreated （MH2） conditions. Through serial dilution and bacteria plate count method, the content of cDNA library and the recombination were calculated. Results showed the content of the MH1 was 4.9×105 and the recombination was 92%, and the content of the MH2 was 7.5×105 and the recombination was 90%. The results of identification of colony PCR showed that the inserts of the two cDNA libraries were basically more than 1 Kb. The two libraries were constructed successfully and could be used for large-scale sequencing. It will provide an effective platform of screening for drought tolerant-related genes and gene clone.3. Large-scale sequencing and EST sequence analysis of two chickpea leaf cDNA librariesAbout 2500 clones from each library were selected randomly for sequencing analysis. Based on IDEG6 online software analysis,92 genes were differentially expressed, and these genes were involved in diverse biological progresses, such as metabolism, transcription, signal transduction, protein synthesis and others. Most of the up-regulated genes were related to drought tolerance, and the down-regulated genes were mainly involved in photosynthesis. The differential expression patterns of 5 functional unigenes were confirmed by quantitative real-time PCR （qPCR）. The results will be helpful in understanding the molecular basis of drought tolerance in chickpea. 4. Gene cloning and expression pattern study of some important genes in S-adenosylmethionine （SAM） metabolic pathwayUnder the basis of 5’EST sequencing, assembling and annotation of the two chickpea leaf libraries, CpSAMs、CpMS and CpSAMDC, which involved in SAM metabolic pathway, were cloned and analyzed by bioinformatics. In order to study the relationship between SAM metabolic pathway and responses of chickpea to drought, the expression patterns of CpSAMs、CpMS、CpSAHH and CpSAMDC were confirmed by semi-quantitative PCR（qPCR）. The results showed the three cloned genes have complete open reading frames. CpSAMs contains two conserved SAMs motifs, CpMS contains Meth_synt₁ and Meth_synt₂ motifs and CpSAMDC contains PEST regions and proenzyme cleavage site motif. The gene expression of CpSAMs, CpMS, CpSAHH and CpSAMDC were up-regulated during the initial stages of drought stress and the three genes also have differentially expression patterns in chickpea roots, stems and leaves. Taken together, it can be speculated that SAM metabolic pathway may be involve in chickpea response to drought stress.