The Expression Of Biosynthesis Genes Of Limonoids And Analysis Of The Accumulation In Tangerine Seed

Limonoids is the major pharmacologically active ingredient compound in Juhe. Topics selected three genes from the downstream stages limonoids compound biosynthetic pathway for depth research - cioning, analysis, expression of ss, se, lgt.analyzed limonoids compound component in stem (phloem), leaves, fruit, seed (germ) and other organs for research limonoids compound biosynthetic machinery and transport law laid the basis.1. ExtractionSample total RNA, functional gene cloning and bioinformatics analysisUsing Omega sample a kit for total RNA extraction and gel electrophorcsis, reverse transcription of RNA purity comply with the requirements. According to the reference sequence homologous gene specific primers were designed to study PCR conditions, PCR products obtained, and the PCR products were sequenced, cloned the ss, se, lgt gene sequences from seed successfully.Key features of the gene sequence alignment and phylogenetic analysis, and its encoded protein sequence alignment right, primary, secondary and tertiary structure prediction, open reading frame identification, hydrophobicity profiles predicted signal peptide cleavage site the aim is to predict the distribution of positions limonoids compound biosynthetic pathway genes and key enzymes in further understanding and analysis, clear structure and characteristics.2. Expression of biosynthesis function gene in limonoids compoundEstablished real-time PCR method for the expression of red orange samples obtained in the stem (phloem), leaves, fruit, seeds (embryo) gene expression of four different organs ss, se, lgt three genes.Three genes expressed mainly trends in seed is ss> se> lgt, the trend of the three genes in the same organs basically the same. Four different organs period of major changes in gene expression maturity, morphogenesis late and dehydrated to a lesser degree of gene expression changes.Seed (embryo) and gene expression was similar pericarp, we are mature after the first decline in three gene expression decreased, there is a period of maximum expression, the expression of the lower end of morphogenesis and dehydration of the three genes. Similar stem (phloem) and gene expression in leaves, the end of the three were morphogenesis gene expression fell and then rose to the highest expression of the existence of a high stage of maturity of the three gene expression increased first and then decreased again rose to the second highest value low expression of the three genes dehydration.3. Active ingredients in different organs accumulated researchUsing UPLC to collect samples for different periods of limonoids, nomilin, one was the determination of Cork, conducted a preliminary study of its accumulation and transfer rule.Limonoids ingredient in seed (embryo) gradually carve three stages accumulation, fell and then built up to the end of the first times a high value, maturity rose to second after the first time the high value decreased, dehydration Fast accumulated the highest value. Limonoids compound content in seeds (embryos) are high and the highest content in the dry period, suggesting that seed (embryo) might be the ultimate storage location limonoids compounds, fully ripe fruit (mid-December) before and after after the harvest is the best medicine of a sharp decline Juhe active ingredient content.Three trends are basically the same ingredients in four different organs, and the size of the order are Limonin> nomilin> Cork ketone. In 130 days of fruit and seed maturation stage, the total content of the active ingredient reproductive organs (seeds) for the increasing trend, the total content of the active ingredient vegetative organs (stems, leaves) and did not change significantly. Presumably to keep the active ingredient may be transported through the vegetative organs of the reproductive organs, and accumulate in reproductive organs.4.Gray correlation between gene expression and limonoids compoundDPS software were used to analyze gene expression and association between active ingredients seed (germ) in limonoids, each active ingredient content nomilin, Cork ketone accumulation lgt genes are greatest contribution, followed ss and se gene. lgt mainly limonoids compound in the biosynthesis of downstream stages, catalytic limonoids compounds into the corresponding glucoside play a role in the process.Stems (phloem), leaves limonoids, each active ingredient content nomilin, Cork ketones accumulate the maximum contribution are ss gene, followed by se and lgt genes. ss and se mainly limonoids compound midstream stage of the biosynthesis of squalene synthetase nomilin process play a role in stem phloem synthetic precursors nomilin, then transport and transformation processes.Stems and leaves of two vegetative organs the total amount of active ingredients subject to the same degree of influence genes; Ju as the main storage site limonoids compound, its chemical composition there is a big difference compared to other parts of the genes affected by the level.