Isolation And Expression Of Two Genes Related To Lignin Synthesis Of Chinese Jujube Fruit

Stone hardening of Chinese jujube （Ziziphus jujuba Mill.） is caused by grit cells, whose major composition is lignin. The study of key genes participating in lignin synthesis is important for revelation of the dynamics of stone development. The cDNA sequences of phenylalanine ammonialyase （PAL） and polyphenoloxidase （PPO） genes related to lignin synthesis from ’Jinsixiaozao’ and ’Wuhexiaozao’ varieties of jujube were cloned in this study. Results show that PAL and PPO genes of these two cultivars are the same. In addition, the study elucidated the gene expression at the transcriptional level during four different development stages of ’Jinsixiaozao’ fruit stone and flesh, and’Wuhexiaozao’fruit stone. The specific results were as follows:1. Cloning and sequencing of Chinese jujube genes related to lignin synthesisThe study obtained the special fragment of target gene with RT-PCR, with the pair of degenerate or specific primers being designed and synthesized according to the conserved domain of the genes. Specific sequence of PAL gene and special sequence of PPO gene were cloned from Chinese jujube fruit by RACE technique. The PAL gene of Chinese jujube was named ZjPAL （HQ616101）, and its cDNA was1955bp long, coding559amino acids,246bp in the3’UTR, and27bp in poly（A） tail. Homologous alignment analysis showed that ZjPAL shared high similarities of92%,91%,90%,89%,89%and88%to the corresponding sequences from Ricinus communis （XP₀02531677.1）, Manihot esculenta （AAK62030.1）, Populus trichocarpa （ACC63889.1）, Vitis vinifera （XP₀02272887.1）, Citrus limon （AAB67733.1）, Pyrus x bretschneideri （ADF59062.1） respectively. The protein predication of ZjPAL shows that there were two endemic protein domains PAL-HAL and Lyase-1-like superfamily which were the prevalent elements of phenylalanine ammonialyase. The PPO gene of Chinese jujube was named ZjPPO （HQ634289）, with its cDNA containing an ORF of1785bp in length, coding594amino acids,145bp in the3’UTR and2bp in5’UTR. Analysis of the putative protein showed that the molecular weight was66.43KDa and the p1was8.23. In addition, homologous alignment analysis revealed that ZjPPO shares high similarities of71%,70%,65%,68%,69%,68%,66%and68%to the corresponding sequences from Cydonia oblonga （ABY84850.1）, Fragaria pentaphylla （ADI44161.1）, Juglans regia （ACN86310.1）, Pyrus pyrifolia （BAB64530.1）, Prunus salicina （AAW65103.1）, Malus x domestica （B21676.1）, Pyrus x bretschneideri （ABS88291.1）, Nelumbo nucifera （ADP89908.1） respectively. The protein predication of ZjPPO shows that there were three endemic protein domains PPO1-KFDV superfamily, Tyrosinase superfamily, PPO1-DWL superfamily which were the major elements of polyphenoloxidase.2. Real-time PCR analysis of ZjPAL and ZjPPOUsing Real-time PCR, the relative expression levels of ZjPAL and ZjPPO were analyzed in four different development stages of’Wuhexiaozao’and’Jinsixiaozao’fruit stone and flesh. The results showed that ZjPAL and ZjPPO in’Wuhexiaozao’and’Jinsixiaozao’fruit stone were consistent in their expression trends, initially increasing, dropping and lastly plateauing. The expression levels of ZjPAL and ZjPPO were highest when the weight of fruit was0.4g, and was lower in’Wuhexiaozao’than’Jinsixiaozao’fruit stone at this stage. There were differences between the ZjPAL expression trends in’Jinsixiaozao’fruit stone and fruit flesh. The ZjPAL expression level in’Jinsixiaozao’fruit flesh was highest when the weight of fruit was0.1g, and then dropped with fruit growth and development. The trend of ZjPPO expression in’Jinsixiaozao’fruit stone was the same as that in fruit flesh, with the highest expression levels occurring when the fruit weight was0.4g and then dropping.