Cloning And Expression Analysis Of Endocarp Genes Related To Lignin Synthesis Of Walnut(Juglans Regia L.)

Walnut kernel phenomenon makes walnut inconvenient postharvest rinsing,transport,storage and other processes within the pericarp is also easily broken,so that cracking rate,the probability of pest fruit rate and seeds contaminated are increased,affecting the appearance quality walnut and sales seriously.Lignin is the main component in the walnut endocarp,so it is significant to research gene of walnut endocarp related to the lignin metabolism and it is a good way to research the mechanism of this phenomenon.In this study,Xinjiang's rare bared-nut walnut ‘Wen 138' were employed for the study of materials and walnut ‘Zhipi' was employed for the study of control.From the endocarp cloned Phenylalanine ammonia-lyase(PAL),4-hydroxycinnamate Co A ligase(4CL)and Cinnamoyl-Co A reductase(CCR)of c DNA fragments of these genes.Expression analysis of three different genes in developmental stages of the hard core of the walnut endocarp by real-time quantitative PCR.1.PAL gene cloning and expression analysis of Walnut(1)‘Wen 138' walnut and ‘Zhipi' walnut PAL gene was cloned by RT-PCR and obtained the c DNA fragment respectively named WJ-PAL and ZJ-PAL.WJ-PAL was contained 600 bp's biggest open reading frame(ORF),can be coded 200 amino acids,and the homology of the black walnut(AJ278454.1)reached 89%.ZJ-PAL was contained 690 bp's biggest open reading frame(ORF),can be coded 230 amino acids,and the homology of the black walnut(AJ278454.1)reached 96%.(2)WJ-PAL and ZJ-PAL deduced amino acid sequence contains phenylalanine ammonia lyase related domains.Both of WJ-PAL and ZJ-PAL contain PAL-HAL nonspecific sites and belonging Lyase_I_Like superfamily.(3)WJ-PAL gene in ‘Wen 138' walnut eight different development period,the relative expression quantity order from high to low is: W726 > W712 > W720 > W621 > W613 > W74 > W628 > W67.ZJ-PAL gene in ‘Zhipi' walnut eight different development period,the relative expression quantity order from high to low is: Z621>Z67>Z712>Z720>Z726>Z613>Z74>Z628.2.4CL gene cloning and expression analysis of Walnut(1)‘Wen 138' walnut and ‘Zhipi' walnut 4CL gene was cloned by RT-PCR and obtained the c DNA fragment respectively named WJ-4CL and ZJ-4CL.WJ-4CL was contained 552 bp's biggest open reading frame(ORF),can be coded 184 amino acids,and the homology of the Betula pendula(KM099195.1)reached 85%.ZJ-4CL was contained 453bp's biggest open reading frame(ORF),can be coded 151 amino acids,and the homology of Betula luminifera(FJ410448.1)and Fagus sylvatica(DQ124216.1)both recched 86%.(2)WJ-4CL and ZJ-4CL deduced amino acid sequence contains 4CL non-specific sites,both of which contain PLN02246,Cai C and AMP-binding functional domains belonging AFD_class_I superfamily.(3)WJ-4CL gene in ‘Wen 138' walnut eight different development period,the relative expression quantity order from high to low is: W726>W712>W720>W613>W621>W628>W74>W67.ZJ-4CL gene in ‘Zhipi' walnut eight different development period,the relative expression quantity order from high to low is: Z712>Z621>Z613>Z74>Z726>Z720>Z628>Z67.3.CCR gene cloning and expression analysis of Walnut(1)‘Wen 138' walnut and ‘Zhipi' walnut CCR gene was cloned by RT-PCR and obtained the c DNA fragment respectively named WJ-CCR and ZJ-CCR.WJ-CCR contains a 954 bp reverse complementary open reading frame(ORF),coded 318 amino acids,ZJ-CCR find out the total of eight ORF,one of the longest ORF of 183 bp,coded 61 amino acids,the shortest is 102 bp,can be encoded 34 amino acids,and the homology of Betula platyphylla(KM505139.1)was 84%.(2)WJ-CCR and ZJ-CCR gene sequences with other plants common functional domains containing FR_SDR_e specific binding sites;PLN02583,3Beta_HSD and PKS_KR nonspecific sites;NADP binding site;PLN02662,Wca G,Hpn A and Epimerase four functional domains;belonging NADB_Rossmann Superfamily.(3)WJ-CCR gene in ‘Wen 138' walnut eight different development period,the relative expression quantity order from high to low is: W712>W74>W726>W720>W628>W67>W613> W621.ZJ-CCR gene in ‘Zhipi' walnut eight different development period,the relative expression quantity order from high to low is:Z712>Z621>Z613>Z67>Z628>Z726>Z74>Z720.