| Peanut (Arachis hypogaea L.) belongs to oil crops and cash crops and is planted in the world. The quality of China’s peanut is good and ranking the first place in the national oil crops export volume. The production of peanut occupies a very important position in China’s economy. At present, up to52.5%of China’s arable land is dryland and more than70%of the peanut acreage suffers from varying degrees of drought. According to conservative estimates, drought can cut more than20%of peanut production in China and has become an important factor in restricting China’s peanut output and improvement of peanut quality. Thence to deepen peanut drought breeding research on the sustainable development of the peanut industry is of great significance in China. We utilized the artificial drought stress method to determine the physiological and biochemical indexes of different peanut genotypes in seedling stage, and laid the foundation of screening drought-resistant peanut varieties. mRNA differential display technology was employed to analysis the genetic differences in peanut seedling stage under drought stress. The screened drought-resistant varieties were used to build the hybrid full length cDNA library of the peanut seedling stage under water stress. Afterwards the library was conducted a preliminary sequencing analysis and laid the foundation of researching and functional analysis of peanut drought related genes. The main results are as follows:(1) Five drought-resistant peanut varieties during seedling stage were screened. Root length, bud length, rooting rate, germination rate and other traits in seedling stage were measured under the use of artificial simulated drought stress. And a comprehensive evaluation was carried out in terms of drought. Thus Guihua99, Huayou89, Hanghua3, Zhanyou58and Shanyouyoul were filtered out. Guihua99which is resistant to drought and Zhongkaihua3which is poor resistance to drought were selected as experimental materials under artificial drought treatment. The biochemical indicators of SOD, POD, CAT and MDA were measured in leaves, and the results showed that the important antioxidant enzyme activity increased faster and had a longer duration in the drought-tolerant peanut varieties under drought stress.(2) mRNA display technology combined46single-lead Scot and44pairs of SRAP primers were applied. Selected out some gene fragments which were differentially expressed. Through NCBI ratio comparison and analysis of collected literatures, four peanut drought-related gene fragments of CDPK, GST, MAPK and SOD were selected to carry out semi-quantitative analysis. And the expression levels of the four gene fragments were proven in roots, stems and leaves in the four peanut varieties. The results were measured in seedling stage under artificial drought stress.(3) RACE technique was used to clone AhP5CS gene full-length sequence. After cloning and sequencing to got3’RACE and5’RACE sequence, ultimately, the full length cDNA sequence of AhP5CS gene was got. The result of ORF prediction showed that AhP5CS gene’s coding sequence (69-2228bp) encoding719amino acids. A detailed bioinformatic analysis of the cloned AhP5CS gene was carried out, and provided an important reference information for the follow-up study of drought-resistant gene.(4) Three drought-resistant peanut varieties were used to construct hybrid full-length cDNA library at seedling stage. Application of "In-Fusion SMARTer Directional cDNA Library Construction technology" was successfully used to construct Guihua99, Hanghua3and Shanyou58three relatively drought-resistant peanut varieties’ hybrid full-length cDNA library in seedling stage. The electrophoresis indicated that the library recombination rate up to98%and the length of inserted fragments between500bp to2500bp. The titer of amplified library was5.81×l011cfu/mL. It indicated that the library could fully meet the needs of a follow-up study.(5) An initial small batch sequencing of the full-length cDNA library was build. The sequencing results showed that the library had a higher rate of full-length sequences and the almost sequences had complete ORF areas. Through analysis of coding gene sequence, the results showed that they had a broader participation in all aspects of life, especially in the expression of induced genes and responsive to external stimuli. This reflects the constructed full-length cDNA library is comprehensive and representative and can be used to select the drought related genes associated with drought in peanut. |
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