Gene Cloning And Functional Research Of ?-Cyano Alanine Synthase Gene In Lathyrus Sativus L.

The planting of grass pea(Lathyrus sativus L.),an underutilized legume that can withstand harsh environmental conditions including drought and flooding,has been greatly limited because of the presence of an endogenous toxin ?-ODAP and low content of cysteine.Beta-cyanoalanine synthase(CASase),which is thought the key enzyme in biosynthesis of ?-ODAP and sulfur assimilatory,might play important roles during the balance between ?-ODAP and sulfur content.Therefore,the cloning and functional investigation of LsCASase gene is vital for the understanding of the regulation of ?-ODAP and further plant breeding of grass pea novel lines with low neurotoxin and high sulfur amino acids content.In this study,cDNA full length of LsCASase gene was obtained first.And then,the sequence was analyzed with the following bioinformatics analysis,tissue-specific expression analysis,RNAi vector construction and plant transformation,etc.Main results were as follows:1.1551 bp full-length cDNA sequence of LsCASase was obtained.The results of bioinformatics analysis indicated that LsCASase encodes 381 amino acids with 1146 bp CDS sequence.CASase,which had CBS-like domain,pyridoxal-5'-phosphate binding site,catealytic residue,located into mitochondria and belonged to Trp-synth-beta II superfamily.The results of multiple alignment and phylogenetic analysis showed that CASase was close to Arabidopsis and soybean CASase.The 3D structure of grass pea CASase was also predicted based on the structure of them.Tissue-specific expression analysis revealed that LsCASase has the highest expression level in the root of 6 DAS,followed by seedlings of 4 DAS,mature leaves and mature stem.The expression level of the seeds germinated 2 DAS was as lower as dry seeds.2.Sense and antisense interference framents were obtained via PCR using the specific primer towards the middle sequence of CASase.And then,BP reaction and LR reaction was conducted to get entry clone(pENTR-CASase)and expression clone(pSGRNAi-CASase)with Gateway cloning technology.The RNAi vector was converted into Agrobacterium GV3101 with freezing and thawing method for LsCASase function characterization.3.In vitro plant regeneration of grass pea was performed from differdnt explants of axillary bud,leaf,and stem explants.And then,Agrobacterium-mediated genetic transformation was conducted to select kanamycin-resistant plantlets.