Cloning And Expression Analysis Of Genes Related With Lignin Synthesis In Pomegranate Seed

Hardness of pomegranate seed was an important index in quality of pomegranate fruit. The hard seed or sofe seed could directly affect the eating feeling and edible portion of the fruit. Sofe-seed fruit of pomegranate was at higher price much more popular in the market at present. Therefore, to study the mechanism of the formation of sofe-seed fruit was significant in pomegranate production.In order to understand the correlation between hardness and total lignin content in pomegranate seed and the expression characteristic of some genes related with lignin synthesis in the seed coat of pomegranate. Selected six kinds of different hardness pomegranate named ’hongyushizi’,’baiyushizi’,’fenpi’,’huiliruanzi’,’mengzitian’ and ’tunisiruanzi’ respectively. Measured the hardness of pomegranate seeds by Texture Analyser. Determinate the total lignin content in pomegranate seed coat by the method of thioglycolic acid. Analyzed the correlation between the pomegranate seed hardness and total lignin content of seed coat. Determinate the total lignin content in pomegranate seed in the decents after the flower full bloom of the 15 days,30 days,45 days,60 days,75 days, 90 days, analysised of the total lignin content changes in pomegranate seeds during growth and development. Cloned the Pg4CL gene and PgCAD gene, which were the important genes of lignin synthesis, by RACE and EST in the seed coat of pomegranate, and cloned partial gene of PgCCoAOMT and PgC4H. Detected relative expression in six different varieties, five different tissue,and six periods with Real-time PCR in pomegranate.The main results had been achieved were as follows.1、The texted the hardness by TA in different mature pomegranate was baiscally identical with the taste hardness. In six varieties, seed hardness and total lignin content in seed were positive correlation, and the correlation coefficient of 0.9224.With fruit growth development,’fenpi’ total lignin content in seeds were on the rise, it was tending towards stability at last.2、Cloned the whole sequence of 4CL gene. The bio-informatics analysis of Pg4CL gene structure showed that cDNA had full length of 2121 bp,the CDS of Pg4CL was 1635 bp,545 amino acids were encoded, including 168 bp of 5’-UTR and 318 bp of 3’-UTR, of which the initiator codon was ATG and the terminator codon was TAA. Homology Comparison showed that the amino acid sequence had high similarity with the corresponding genes from Vitis vinifera, Citrus sinensis and Populus trichocarpa at 92.83%,92.62% and 92.07%, respectively.3、Cloned the whole sequence of CAD gene. The bio-informatics analysis of PgCAD gene structure showed that cDNA had full length of 1226 bp,the CDS of PgCAD was 981 bp,327 amino acids were encoded, including 51 bp of 5’-UTR and 194 bp of 3’-UTR, of which the initiator codon was ATG and the terminator codon was TGA. PgCAD and Pg4CL genes sequence checked in the Genebank, landing AFU90743.1 and AFU90744.1 respectively. Homology Comparison showed that the amino acid sequence had high similarity with the corresponding genes from Rosa rugosa, Populus trichocarpa and Prunus mume at 93.46%,93.49% and 92.87%, respectively.4、In different varieties of pomegranate seeds, Pg4CL, PgCAD, PgC4H and PgCCoAOMT four genes were expressed,the’tunisiruanzi’of four genes expression quantity were relatively high, relative expression of several other varieties of seed coat total lignin content is relevant.5、In different tissues of the pomegranate, the amount of lignin synthesis of related enzyme gene expression existed differences between tissues. Pg4CL gene quantity expression was highly in peel, followed by leaves and stems, flowers and seeds in the quantity expression was low. PgCAD genes quantity expressed highly in seed, followed by flowers, were not expressed in the leaf and the peel. PgC4H genes quantity expressed highly in leaf, the second order for peel and flowers, quantity expressing was low in seed and stem t. PgCCoAOMT genes relative expression is higher in the flowers, followed by leaves and stems, and expressed in skin and seed was low.6、Different varieties of 4CL, CAD, C4H and CCoAOMT of gene expression had no obvious correlation between the content of total lignin.At different times after flower blooming. They were relative expression of the genes in different peak period, the relative expression of CCoAOMT and C4H gene peak for the 45th day after flower blooming.4CL gene and CAD reached the height of the relative expression of for 60 days after the flower blooming.Four genes expression quantity was different between different varieties.