Construction Of CDNA Library From Leaves Of Solanum Torvum & Genetic Transformation Of Salt Tolerance Related StBADH Gene In Tomato

Wild eggplant (Solanum torvum Swartz), originated from Indonesia (Java island) has high resistance to diseases and stresses, which was introduced into Japan in 1986. After hybrid breeding for many years, a new salinity-tolerant rootstock variety,’Torvum Vigor’, had been bred out. The objectives of this research were to construct a cDNA library from the leaves of salinity-tolerant wild eggplant variety,’Torvum Vigor’, and then expressed sequence tag (EST) analysis with bio-informatics software, and a new salt tolerance related gene named StBADH was cloned by RT-PCR technique. An herbicide-resistant plant expression vector pCAMBIA3301-StBADH-T800 was constructed, and then was transferred into tomato cultivar, ’Jinpeng No.l’. Finally, we obtained transgenic tomato plants which can resist herbicide. The main results are as follows:1. cDNA library from the leaves of wild eggplant variety,’Torvum Vigor’ was successfully constructed by a switching mechanism at 5’-end of RNA transcript (SMART) approach and a long-distance PCR (LD-PCR) technique using pBluescript II SK (+) vector. The titer of the primary cDNA library was 3.6×106 cfu’mL-1 and that of the amplified library was 1.2 ×1010 cfu’mL-1. Gel electrophoresis results showed that most of the cDNA inserts ranged from 0.40 to 2.5 kb, with a recombination rate of 99%. These results showed that this library was highly quality, which is suitable for the isolating and screening target genes.2. A total of 427 randomly selected positive clones from the constructed cDNA library of wild eggplant leaves were sequenced. After removing the unsuccessful reads,364 datasets were obtained and have been submitted to the NCBI Nucleotide Sequence Database (GenBank accession numbers:JK265131-JK265494). Among the 364 submitted sequences,74.72% of them contained full-length coding regions. BLASTX analysis revealed that 62.4% of the EST possessed homology to reported proteins of other organisms, nine ESTs that might be responsible for the salt tolerance traits (five salt tolerance related genes).3. The salt tolerance related gene StBADH (GenBank accession number:JN812057) was cloned from the leaves of wild eggplant variety, Torvum Vigor’by RT-PCR technique. The gene nucleotide sequence size was 1518 bp, contained a complete open reading frame, encoded 505 amino acid residues, molecular weight was 55.9 kDa, isoelectric point was 5.42, its amino acid sequence homology was 96% with the tomato. The phylogenetic tree analysis revealed that wild eggplant betaine aldehyde dehydrogenase and tomato with the same family cluster.4. The pT800 plasmid (containing the CaMV35S promoter, pUC18 Polylinker multiple cloning site, Nos terminator) and plant expression vector pCAMBIA3301 (containing bar) by restriction enzyme Hind III and EcoR I double digestion, respectively, constructed the intermediate vector pCAMBIA3301-T800. Then StBADH and intermediate vector pCAMBIA3301-T800 by restriction enzyme Sma I and Xba I double digestion, respectively, the successful construction of the herbicide-resistant plant expression vector pCAMBIA3301-SrBADH-T800, and this plasmid was transferred into Agrobacterium tumefaciens LBA4404.5. Using tomato (Lycopersicon esculentum Mill.) cultivar,’Jinpeng No.1’hypocotyl and cotyledon as explants, study on the effects of different hormones correlated in adventitious shoot regeneration, and rooting. Meanwhile, using Agrobacterium-mediated method genetic transformation of the herbicide-resistant plant expression vector pCAMBIA3301-StBAD#-T800 in tomato (Lycopersicon esculentum Mill.) cultivar, ’Jinpeng No.1’. The results showed that:the 10 d seedling age hypocotyl and cotyledon in MS+ 0.2 mg-L-1 ZT+ 1mg·L-1 IAA of shoot induction medium develop the highest frequency of shoot regeneration, hypocotyl was 83.3%、cotyledon was 75.6%. MS+ 0.5 mg-L"1 IAA was the optimum rooting medium,7 d after rooting was 100%. Shoot regeneration medium supplemented with bialaphos of the optimum screening concentration is 2 mg·L-1, and rooting medium supplemented with bialaphos of the optimum screening concentration is 1 mg·L-1, carbenicillin inhibitory concentration is 500 mg·L-1. As a result, the average genetic transformation efficiency of 2.5%(2/80). Finally, we obtained two To transgenic tomato plants which have shown resistance Basta, and screened by the histochemical GUS assay, leaves and roots of two transgenic plants GUS chemical strain showed expression blue band, PCR detection of StBADH、bar and GUS were positive.