Cloning And Expression Analysis Of Endocarp Genes Related To Lignin Synthesis Of Bared-nut Walnut

Xinjiang is the birthplace and one of the main producing areas of China's walnuts?Juglans regia L.?is also.In order to understand the mechanism of the endocarp of walnut in Xinjiang,the cDNA sequence of C3 H,C4H,CCoAOMT and F5 H gene were cloned from 'Wen 138' and'zhipi' walnut by using RT-PCR.They were named WJ-C3 H,ZJ-C3 H,WJ-C4 H,ZJ-C4 H,WJ-CCoAOMT,ZJ-CCoAOMT,WJ-F5 H and ZJ-F5 H,and their sequence characteristics,structural characteristics and evolutionary relationship were analyzed;Through real-time fluorescence quantitative PCR,the relative quantitative expression analysis was carried out in 9periods?44,51,58,65,72,79,86,93 and 100 d after flowering?of the endocarp hardening process,and the spatial and temporal expression characteristics of the hard core stage were obtained.The main research results are as follows:The cDNA sequence of WJ-C3 H gene contains 702 bp ORF,encoding 233 amino acids,total number of atoms is 3694,molecular weight is 26.16 kDa,theoretical isoelectric point is6.55;the ZJ-C3 H gene cDNA sequence contains 636 bp ORF,encoding 211 amino acids,total number of atoms is 3352,the molecular weight is 23.77 kDa and isoelectric point is 5.97.There are one CypX-specific binding sites in the deduced amino acid sequences of WJ-C3 H and ZJ-C3 H genes,and three non-specific binding sites including PLN02687,p450 and P450_cycloAA₁,all belonging to the p450 superfamily.The cDNA sequence of WJ-C4 H gene contains 931 bp ORF,encoding the protein 305 amino acids,total number of atoms is 5031,molecular weight 35.64 kDa,theoretical isoelectric point is 7.75;The ZJ-C4 H gene contains 952 bp ORF,encoding 312 amino acids,total number of atoms 5130,molecular weight 36.38 kDa,the theoretical isoelectric point is 8.67.The predicted expression of the encoded protein indicated that the deduced amino acid sequences of WJ-C4 H and ZJ-C4 H genes had two specific binding sites,p450 and CypX,and one non-specific binding site of PLN02394,which belonged to the p450 superfamily.The cDNA sequence of WJ-CCoAOMT gene contains 672 bp ORF,encoding 224 amino acids,total number of atoms is 3449,molecular weight is 24.20 kDa,and theoretical isoelectric point is 11.50.The cDNA sequence of ZJ-CCoAOMT gene contains 672 bp ORF,encoding 224 amino acids,total number of atoms is 3468,molecular weight is 24.44 kDa,and theoretical isoelectric point is 11.02.Predictive analysis of the encoded protein indicated that the two deduced amino acid sequences contained three non-specific binding sites,PLN02589,Methy1transf₃ and YrrM,belonging to the AdoMet_MTases superfamily?including a YrrM superfamily in this interval?.The cDNA sequence of WJ-F5 H gene contains 535 bp ORF,encoding 176 amino acids,total number of atoms is 2796,molecular weight is 19.89 kDa and theoretical isoelectric point is4.66.The cDNA sequence of ZJ-F5 H gene contains 535 bp ORF,encoding for 176 amino acids,total number of atoms is 2810,molecular weight is 19.98 kDa,theoretical isoelectric point is4.73.TThe WJ-F5 H gene deduced amino acid sequence has a specific binding site CypX and PLN02183,p450,P450_cycloAA₁ three non-specific binding sites;ZJ-F5 H gene deduced amino acid sequence exists in the PLN02183,p450,CypX,P450_cycloAA₁ four non-specific binding sites Point,the deduced amino acid sequences of WJ-F5 H and ZJ-F5 H genes belong to the p450 super family superfamily.The results of real-time PCR showed that the four key genes of WJ-C3 H and ZJ-C3 H,WJ-C4 H and ZJ-C4 H,WJ-CCoAOMT and ZJ-CCoAOMT,WJ-F5 H and ZJ-F5 H were hardened in the endocarp of walnut.The relative expression levels of C3 H genes in different periods showed a trend of “?”,the 'zhipi' walnut showed “M”,and the C4 H and F5 H genes were 'wen138'.The relative expression changes of 'zhipi' walnuts showed an "N" change trend,the CCoAOMT gene 'Wen 138' showed an "N" change trend,and the 'zhipi' showed a "M" change trend;The expression of the gene in the endocarp was 72-79 d after flowering,and the overall change trend had a "V" shape change region.The three genes WJ-C3 H,WJ-CCoAOMT and WJ-F5 H were spent.The expression of 51⁶5d was significantly lower than that of ZJ-C3 H,ZJ-CCoAOMT and ZJ-F5 H.The expression levels of WJ-C4 H,WJ-CCoAOMT and WJ-F5 H were significantly higher than those of ZJ-C3 H,ZJ-CCoAOMT and ZJ-F5 H at 79-93 days after flowering.