Cloning And Expression Of Gene Encoding Glycilin From Pigeonpea [Cajanus Cajan (L.)Millsp.]

Pigeonpea(Cajanus cajan. Millsp) is the sixth largest edible beans, the only woody edible beans in the world, and has been an important human and animal source of vegetable protein with high protein, drought and barren resistance, and good at fixing Nitrogen of air. It was distributed in tropical and subtropical areas of the world. It can be used mainly for edible and also be using as a high-quality protein green fodder, soil and water conservation, and cover crops etal. As all are known, it is particularly lacking of vegetable protein in the tropical and subtropical areas. Pigeonpea is an important rich in protein crops and it has great significance to excavate the gene resources of its excellent protein in pigeonpea breeding and other tropical and subtropical rich in high protein crop breeding.According to homology sequence from Glicine max, Pisum sativum, Phaseolus vulgaris, Lupinus angustifolius, Arachis hypogaea and so on, by means of designing degenerate primers, Reverse transcription Polymerase Chain Reaction(RT-PCR) amplification and agarose gel electrophoresis, Blastn and Blastp analysis tools of NCBI database, a special Complementary DNA(cDNA) fragments from the pigeonpea material has been gotten and contains833bp at the first.The full-length cDNA encoding Pigeonpea glycinlin(PPGLY) was cloned from pigeonpea by RT-PCR,5'and3'Rapid Amplification of cDNA Ends(RACE) technology. It contained1595bp. By Open Reading Frame(ORF) software analysis, it found that the cDNA contained a1551bp, ORF (16-1566bp), encoding516amino acids. It also showed that the gene Coding sequence (CDS)of A+T content is50%, C+G content is also50%by statistics analysis. The deduced amino acid sequence of PPGLY molecular weight is57.718kDa, and its isoelectric point (pI) is4.68. It also showed that strong basic amino acids (K, R)48, accounting for9.30%; strong acidic amino acids (D and E)84, accounting for16.28%; polar amino acids (N, C, Q, S, T, Y)153, accounting for29.65%; hydrophobic amino acids (A and I, L, F, W, V)144, accounting for27.91%through amino acid composition analysis.The deduced amino acid sequences of PPGLY show hight identity (83%,79%,77%,75%,67%,73%,65%and65%) with Glycine max (1303273A), Glycine max (CAA55977.1), Lupinus angustifolius (AEB33710.1), Lotus(CAR78991.1), Arachis hypogaea (AAU21493.1), Phaseolus vulgaris (ADR30064.1), Vicia fatal(CAA83674.1), truncatula Medicago protein (XP₀03590690.1).By quantitative Real-time RT-PCR in different growth stages (vegetative, flowering and maturity),the relative expression levels of PPGLY gene of the root, stem, leaves, flowers and seeds show that the expression level of the root in vegetative phase is minmum and the expression level of mature seeds is maximumThe expression level of growth period is that:Vegetative<Flowering<Maturity, vegetative, and was gradually increased; it exists some differences in different tissues of differen growth period also:①vegetative phase: Root<Stem<Leaf;②Flowering phase: Root<Stem<Leaf<Flowers;③The maturity phase: Root<Stem<Leaf<Flower<Seed.