Cloning, Function Analysis And Single Nucleotide Polymorphism Of Common Bean (Phaseoleae Vulgaris L.) P5CS Gene

Common bean is one of the most important edible food legumes in the world. Drought and soil salinity are the most serious environmental stresses that limit common bean growth and productivity. Therefore, they are very important to study stress tolerance mechanism and utilize genes involved in crop drought and salt tolerance for the improving common bean variety. We have cloned two P5CS genes, PvP5CS1 and PvP5CS2 using a candidate gene approach from common bean. Real-time quantitative PCR was used to examine their responses to different stresses. The genes have also been transformed to Arabidopsis and tobacco plants, respectively. Single nucleotide polymorphism (SNP) distribution of PvP5CS2 had been analyzed using direction sequencing method. The results are mainly as follows:1. Two full-length cDNA denominated PvP5CS1 and PvP5CS2 forΔ1-pyrroline-5-carboxylate synthetse (P5CS), an enzyme involved in the biosynthesis of proline, were cloned from common bean. The length of PvP5CS1 nucleotide sequence was 2.246 kb with 2.151 kb open reading frame. The cDNA sequence of PvP5CS2 was 2.340 kb, containing an open reading frame of 2.148 kb, which was separated by 19 introns in DNA sequence. Sequence analysis showed that PvP5CS1 and PvP5CS2 shared 95.1% and 82.6% homology respectively in nucleotide sequence and had 93.2% and 79.9% identity respectively in amino acid sequence with that of VaP5CS. Two PvP5CS proteins showed the conserved Glu-5-kinase and GSA-DH domains with other plant P5CS proteins. They also had putative ATP and NAD(P)H-binding sites and two Leu-rich regions.2. The expressions of the two PvP5CS genes were significantly induced by drought, salt (200 mM NaCl), and cold (4°C) stresses. Drought stress induced two genes up-regulated expression to the maximum at 4th day, which was earlier than the proline accumulation. Under salt stress, the mRNA levels of two genes reached to the maximum at 2 h in leaves and 6 h in roots, while the peak of proline accumulation appeared at 9 h. Cold stress inhibited the expression of PvP5CS1 in root, but caused it accumulation in leaves. PvP5CS2 mRNA levels rapidly enhanced to maximum at 2 h in leaves and roots of common bean seedlings treated with cold stress. Under cold stress condition, proline content was slowly enhanced to the maximum at 12 h in leaves and at 24 h in roots.3. Introduction of PvP5CS1 gene into Arabidopsis resulted in accumulation of proline and improved the transgenic plants tolerance to drought and salt stresses. Under unstressed conditions (CK), 150 mM mannitol-, 250 mM mannitol- and 150 mM NaCl-stress, the proline contents in transgenic plants were 1.38, 2.68, 1.30 and 1.30 times of wild type plants. Seed germination rate of wild plants was lower than transgenic lines treated with 150 mM NaCl and 150 mM mannitol. The transgenic lines had longer root and lower degree of cell damage than wild plants under osmotic and salt stress conditions. The living rates of transgenic line seedlings were all higher than wild plants treated with 300 mM NaCl for 15 days or withholding water for 30 days.4. Overexpression of PvP5CS2 gene enhanced the drought tolerance of transgenic tobacco plants. Under water stress condition, the leaf proline content, wilt leaf account and leaf relative water content in transgenic plants were 2.3, 0.88 and 1.26 times of wild plants, respectively.5. 63 SNP sites and 224 InDels were found in PvP5CS2 gene among 27 accessions. There were 18 SNPs distributing in coding region and 45 SNPs distributing in no-coding region. All InDels were distributed in no-coding regions. PvP5CS2 was a conservative gene underwent strong negative selection pressure (Ka/Ks<0.5) in common bean evolution. The nucleotide diversity (π) of the gene in 27 common beans is 0.00483, which was higher than that in Andean gene pool (π=0.00107) and Middle American gene pool (π=0.00144). The single nucleotide polymorphism of PvP5CS2 was not associated with the drought tolerance of common bean, but closely correlated with common bean origin.6. An amplification fragment length polymorphism marker, Pv97, developed based on an InDel between G19833 and DOR364 could be credibly used for the tracing common bean gene source. PvP5CS2 gene was mapped on the b01 chromosome of common bean.7. Transient expression of GFP/PvP5CS1 and GFP/PvP5CS2 in onion epidermal cells respectively showed that these two proteins were distributed in the nucleus and at the plasmalemma.