Cloning And Expression Analysis Of Anthocyanin Biosynthesis Genes Of Anthurium Andraeanum L.

Anthurium andraeanum L. belonging to the Araceae Anthurium, is native to tropical regions of Central and South America. Its unique flower spathe with brightly gorgeous and rich colors has high ornamental value. Color is one of the most important traits of ornamental plants and also is one of the targets of genetic breeding. The breeding of Anthurium overseas was mainly through the screening of wildlife resources, while there is no wild populations’ distribution of anthurium in China, cultivars are drawn from abroad, even the genetic background of Anthurium is very complicated, therefore the application of molecular biology techniques for the introduction Anthurium varieties of genetic improvement to cultivate new varieties with independent intellectual property rights, has good prospects in the development of flower breeding work. In this study, the spathe of anthurium cultivars ’Dakota’ is divided into eight stages(S1-S8) from extracting to aging. Full-length cDNA sequences of AnC4 H and AnCHI genes were cloned through the results of transcriptome sequencing which expressed differentially in the anthocyanin biosynthesis pathway by using the rapid amplification of cDNA ends(RACE) method. Analyzing the relationship between gene expression and anthocyanin content to further improve and provide research basis of Anthurium anthocyanin metabolic pathway, and it may be lay the foundation of modified Anthurium flower color artificially to to further enhance its ornamental and commercial value. The main results are as follows:1. By using visual color measurement, RHSCC colorimetric to analysis variety of phenotype and measure anthocyanin content of anthurium spathe color during different period flower development. We found that the spathe color during S1-S5 period is in red lines with higher total anthocyanin content and has upward trend; it began to turn green in period of S6 with a sharp decline in total anthocyanin content; the period S8 growed into whole of green with the lowest content of anthocyanin.That is to say the color phenotypes of anthurium spathe are directly relative to the anthocyanin content which is higher in initial stage of spathe development than adult.2. Two structural genes were preliminary screened from the metabolic pathway of anthocyanin in anthurium spathe based on the results of the laboratory transcriptome data, as was called cinnamic acid4-hydroxylase(C4H) and chalcone isomerase(CHI). Full-length cDNA sequences were isolated by using the RACE method, which named AnC4 H and AnCHI(accession number KU356776).3. The expression patterns of the key gene AnC4 H and AnCHI in eight stages of spathe, roots,stems, leaves and spadixes were analyzed by qRT-PCR, the results showed that they were detected in various tissues, mainly in the spathe where they had the highest expression of the third growth period(S3), and the low expression in roots, stems and spadixes. And during the period 1-4 of spathe the expression was significantly higher than the period 5-8. This result indicates that AnCHI and AnC4 H gene have high expression levels in the early development of spathe and reducing the expression levelsafter maturity. While taking advantage of the correlation analysis between anthurium anthocyanin content and gene expression was 0.90 by software SPSS Statics.4. The protein of gene AnCHI and AnC4 H were localized in the cell membrane and plastids of onion via subcellular localization technology. The protein of gene AnCHI was a kind of soluble protein mainly expressed in the supernatant of E. coli by prokaryotic expression technology, which can provide theoretical basis for the subsequent research of gene clone and interaction with other proteins.