Clone And Expression Analysis Of Related Genes Of Benzyl Glucosinolate Biosynthetic Pathway In Carica Papaya

It is reported that a potential cancer-prevention and anti-cancer thioglycoside in recent years studies, benzyl glucosinolate (BG) exists in all tissues of papaya fructus except in the mature pulp of fruit. Benzyl glucosinolate (BG) can be hydrolyzed into benzyl isothiocyanate(BITC) by myrosinase. Cancer has been the number one killer of human health. The world health organization pointed out that at present, about10million people have been suffered from cancer in the world every year, of which more than6million people died.The cancer prevention and anti-cancer activities of benzyl isothiocyanates (BITC) toward mammals have been gained recognition in medical field. Carica papaya L., a perennial ever-green herbaceous plant belongs to genus Carica of family Caricaceae, has a short growth cycle. Its fruit is a kind of tropical fruit of high nutrition value. Further research and development of high value-added products helps to promote the development of Papaya industry.The "sunrise" breed of papaya was chosen to study in Papaya base in Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. The HPLC assay method of BG and GC assay method of BITC in papaya were established firstly and the content of BG and BITC in each part of tissue were determined. Referred to the biosynthetic pathway of BG in arabidopsis and according to the reported genome sequence of papaya, we studied the related enzyme genes in the biosynthesis of BG in papaya, which laid a certain foundation for the exploitation and gene regulation of BG in Papaya. The main research results are as follows:1. The assay method of BG determined by HPLC and the content determination of BITC by GC were respectively established through systematic methodology and the content of BG and BITC in the root, stem, leaf, flower, fruit and seed were determined in Papaya. The content of BG in the seed of Papaya is the highest of all (4.7μmol.g-1), the content in the radicle and young stem is the second, the young leaf and flower ranks third, and there almost no BG determined in pulp. BITC was detected only in the seed and flower, and the content of BITC in the seed is also the highest of all tissues, which reaches up to0.605mg·g-1. And through the experiment in vitro we confirmed that BITC has better inhibition action to proliferation to human lung cancer cell line H69.2. Six genes in the biosynthetic pathway of BG in Papaya were cloned. The all-length primer was designed by comparison and by means of the whole genome sequence (ABIM01000001-ABIM01047483), the genes were cloned with the cDNA of young leaf as template and then the sequence were blast to infer the functions of genes. The obtained6genes were as follows:among the five steps of synthetic pathways, phenylalanine is oxidised into phenylacetaldoxime, and two enzyme genes were cloned in the first step, which were named CP-CYP79A2.1and CP-CYP79A2.2; only1enzyme gene was cloned in the other4steps, which were named CP-CYP83B, CP-C-S,CP-UDP-T and CP-ST5a, respectively.3. The expression level of the6genes and the reported myrosinase (tgg1and tgg2) gene in different tissue parts of Papaya were studied using real-time quantitative PCR technology. The expression level of the6genes in the radicle and young stem is relatively higher, the levels in the leaf and flower are relatively lower, the level in the seed is the lowest of all and the level in pulp is a little higher than that in the seed. The expression level of tggl is relatively high in the stem, the levels in the flower and seed are the next, and the levels of tggl in the root, leaf and pulp are the lowest. The expression level of tgg2in the root is far higher than that in other tissues. The expression level of the six genes in the synthesis of BG in papaya falls in downtrend on the whole according to the different parts of root, stem, leaf, flower, pulp and seed in order. However, the results of the content of BG determined indicated that the content of BG was the highest in the seed. Referred to the literature, we can infer that the reason is that after the thioglycoside was synthesized in the pulp, it was transferred into the seed and reserved in it.4. By means of protoplast and RNAi technologies, the constructed vector containing ihpRNA hairpin (target gene:CP-CYP83B) was transferred into protoplast through PEG mediation. Then they were transferred into the protoplasts of mesophyll in papaya and cultured for48h. The expression level of CP-CYP83B gene is obviously declined and the content of BG in the protoplast is significantly reduced, which indicate that target gene CP-CYP83B is the related gene in the biosynthesis of BG and provide a technology platform for studying gene functions of papaya by protoplast.