Study On Amino Acid Metabolism And PLCP Gene Function In Grape Seed

Programmed cell death,protein degradation are involved in grapevine(Vitis vinifera L.)seed development and abortion,presumably leading to amino acid level change and nitrogen reuse.To explore the genes functions of PLCP involved in protein hydrolysis and genes involved in Asn and Gln metabolism of high N/C during grape seed development and abortion,this study determined the contents of main amino acids in seeds,identified Vvi PLCP,Vvi Cystatin,Vvi ASN and Vvi GS family members,analyzed the evolution and expression patterns.Moreover,the preliminary functions analysis of Vvi ALP and Vvi CYS6 was carried out.The results were as follows:1.Using high performance liquid chromatography-mass spectrometry(HPLC-MS),the contents of 16 kinds of amino acids in grape seeds were determined.The results showed that each amino acid in seeded and seedless grapes were obviously different.The content of Asn significantly increased in seedless grape since the early ovule development,more than in seeded grape,and decreased sharply at the middle and late stage.It was suggested that seed abortion might start since the early ovule development,endosperm protein degradated and nitrogen was mainly stored and transported in the form of Asn.2.Eight Vvi Cystatin and 22 Vvi PLCP family genes were identified and analyzed by bioinformatics.The gene expressions were analyzed through q RT-PCR.Vvi Cystatins and Vvi PLCPs were both conserved,respectively divided into 3 and 9 groups with specific characteristics.The expressions patterns of Vvi Cystatins and Vvi PLCPs were obviously different in various tissues and seeds of various grape varieties.The vast majority of the genes expressed more in ‘Flame seedless' than in ‘Pinot Noir' from 10 or 15 DAF.Different genes responded to hormones with various patterns.Therefore,it was speculated that seed abortion might start from the early ovule development.Vvi PLCPs and Vvi Cystatins were involved in different hormone signal pathways,and they might coordinately regulate the degradation of seed proteins,the remobilization of amino acids,and the regulation of seed development and abortion.3.Vvi ALP and Vvi CYS6 were cloned,and the interaction between Vvi ALP and Vvi CYS6 was verified by yeast two-hybrid assay and bimolecular fluorescence complementary.The overexpression vectors were constructed and expressed in tobacco.It was found that Vvi CYS6 was involved in the resistance to phytophthora capsicum,and might inhibit Vvi ALP resulting in reduced disease resistance.4.Two Vvi ASN and five Vvi GS family genes were identified,cloned and analysed by bioinformatics.The gene expressions were analyzed through q RT-PCR.Vvi ASNs and Vvi GSs were highly conserved,and divided into two groups.The expression patterns of Vvi ASNs and Vvi GSs were tissue-specific.Most genes expressed more in seedless grapes than in seeded grapes,and the expression of Vvi ASN1 was positively correlated with Asn content.Moreover,Vvi ASNs and Vvi GSs responded to hormones with different patterns.It was speculated that Vvi ASNs and Vvi GSs might participate in the amino acid metabolism of seed abortion through different hormone signaling pathways,and nitrogen might be stored mainly in the form of Asn.