Cloning And Functional Analysis Of Stcyp707as Genes From Potato

Potato(Solarnum tuberosum L.) is ranked4th in crop production following rice, wheat and maize. Potato tubers are propagation organ as well as product organ. Studying regulatory mechanism of potato tuber formation does not only have a theoretical value but also significance for improving potato production. Regulation of potato tuber formation is a complex biological process affected by internal factors and external factors. The external factors include light, temperature, nitrogen level, etc. and the internal factors include phytochrome, transcription factors, plant hormones, etc. CONSTANS (CO) is a transcription factor and it plays a key role in the flowering induction of plants. Recently, our research found that the StCOL anti-sense inhibition of transgenic potato plants regulated the expression of StCYP707A gene, a key enzyme gene in the decomposition of ABA-8’-hydroxylase (8’-hydoxylase). In this study, StCYP707Al, StCYP707A2, and StCYP707A3gene were cloned from potato and StCYP707Al over-expression and anti-sense inhibition transgenic poatato plants were constructed. In addition, the function of StCYP707Al in potato was analyzed. The main results are as follows:1) StCYP707Al gene was isolated from the potato cultivar "Desiree" by RT-PCR. StCYP707Al gene constituted by six introns and seven exons. The cDNA sequence contained a1407bp open reading frame encoding a469amino acid, with an MW of53417.2Da, a PI of8.90. StCYP707Al protein contained the Cytochrome P450cysteine heme-iron ligand signature, accorded with P450family of single oxygenase characteristics. StCYP707Al and other homologous protein amino acid sequence alignment showed that StCYP707Al was close with AtCYP707A2. Tissue expression analysis showed that the highest expression level of StCYP707Al was observed in the leaves, then in roots and stem. In addition, StCYP707Al could be induced by low temperature, drought, salt treatment, V. dahliae infection and SA.2) Potato plants were transformed with over-expression vector\304-StCYP707Al and anti-sense inhibition vector1304-StCYP707Al-F by Agrobacterium mediated method. Four plants of over-expression and nine plants of anti-sense inhibition were obtained by PCR test. Tuber yield analysis showed that over-expression of StCYP707Al in transgenic plants increased significantly yield compared with the control, while anti-sense inhibition caused a decrease in tuber yield.3) StCYP707A2was isolated from the cultivar "Desiree" by RT-PCR. The StCYP707A2gene contained an ORF of1425bp encoding a protein composed of475amino acid residues, with a molecular weight of54271.9Da and isoelectric point for9.31. StCYP707A2protein contained a Cytochrome P450cysteine heme-iron ligand signature and accorded with P450family of single oxygenase characteristics. Sequence alignment of StCYP707A2and other homologous protein amino acid showed that StCYP707A2was close with S1CYP707A1. Tissue expression analysis showed that StCYP707A2was expressed in stem, leaves, but rarely be detected in roots. And StCYP707A2could be induced by low temperature, drought, salt treatment, V. dahliae infection and SA.4) StCYP707A3was isolated from the cultivar’"Desiree" by RT-PCR. StCYP707A3gene constituted by five introns and six exons. The cDNA sequence contained an1329bp open reading frame encoding a protein of443amino acid residues with molecular weight to50524.0Da and isoelectric point of8.95. StCYP707A3contained a Cytochrome P450cysteine heme-iron ligand signature and belonged P450monooxygenase family. Sequence alignment of StCYP707A3and other homologous protein showed that StCYP707A3and AtCYP707A2were in the same cluster. Tissue expression analysis showed that StCYP707A3was expressed in roots, stems and leaves. It could also be induced by salt treatment, V. dahliae infection and SA.