EaC4H Gene Cloning And Allelopathy Related Gene Functional Analysis From Eupatorium Adenophorum

Eupatorium adenophorum is the first one as the most ecologically significant and potentially destructive species for China as a kind of noxious invasive alien weed and so it has caused serious loss in economy, ecology and society, was introduced into China in 1940s. Now, most of the study was focused on the invasion and dissemination patterns of E. adenophorum, its physiological ecology, prevention and utilization of the weed. Those allelochemicals of E. adenophorum play an important role in process of biological invasion. Its mechanism probably influences many aspects in plant, such as photosynthesis, various metabolisms, root growth and metal elements absorption. Little research was reported about reguLation mechanism of allelochemical in the secondary metabolic pathways, and the work of our research involves. The gene was cloned and the function was analysised in Eupatorium adenophorum in this study to provide a theoretical basis for the all-sided control of this malignant weed through the research in metabolic pathway related to allelochemical. By the study, we try to find out the coding gene of key enzyme which is related with the metabolism of allelochemical and clone some novel genes which can be used in agricuLtural production. The resμLts are as follows:The Cinnamate-4-hydroxylase gene (EaC4H) of E. adenophorum was cloned by using rapid-amplification of cDNA ends(RACE) method (GenBank ID:KF751239). The 1518 bp open reading frame (ORF) of EaC4H was deduced to encode a protein of 505 amino acids residues. Like other C4Hproteins, the predicted EaC4H polypeptides contained conserved domains of cytochrome P450. Using the sequence of 3’UTR in EaC4H gene as primers, the real-time quantitative PCR result showed that the expression level of EaC4H gene was up regulated when induced by HHO.Southern blot analysis indicated that at least five copies of EaC4H existed in the genome of E. adenophorum. Subcellular localization revealed the nuclear-localized EaC4H-GFP fusion protein in onion epidermal cells.We constructed the over-expression vectors of EaCHS and EaC4H genes respectively, and the RNA interference vectors of the genes were constructed respectively too, and then transferred them into NC89 tobacco by the method of leaf disc infection to search the gene function.We determinated the genes expression related allelopathy metabolism and the content of total phenol and flavonoids in EaCHS transgenic tobacco by Real-time PCR (RT-PCR). Overexpressing of EaCHS in tobacco resulted in increased expression of some related genes and production of downstream flavonoid and the total phenol in phenylpropanoid pathway. However, heterologous silencing of endogenous CHS in tobacco by conserved EaCHS fragment led poles apart in results.The genes expression related allelopathy metabolism and the content of total phenol and flavonoids in transgenic tobacco were determinated by Real-time PCR (RT-PCR). Heterologous silencing of endogenous C4H in tobacco by conserved EaC4H fragment resulted in reduced expressions of key enzyme genes and production of downstream flavonoid in phenylpropanoid pathway. Intriguingly, the similar effects were observed in transgenic tobacco plants of overexpressing EaC4H. Altogether, our results indicate that disturbed expression of C4H in plants leads to relatively low expression levels of key enzyme genes and accumulation of flavonoid involved in phenylpropanoid metabolism.Salt and drought stressis are the major abiotic stress in agricμLture world-wide, which affect almost all aspects of plant development, including germination, shoot growth and RSA development. In terms of response to abiotic stress, these physiological indexes such as chlorophyll content, MDA and POD content, SOD activity and CAT activity had been widely used in related studies announced previously.First, we tested whether EaCHS was related to these physiological indexes under salt stress condition. The results showed the germination rate of transgenic tobacco plants of overexpressing EaCHS was higher than EaCHS RNAi line and wild type (WT) under the salt or drought stress. The results of chlorophyll content, MDA and POD content, SOD activity and CAT activity in transgenic tobacco had further proved that the overexpressing EaCHS plants have stronger resistance than the others. All these physiological results manifested that EaCHS gene is involved in reguLating the plant tolerance to salt or drought stress.Second, we tested whether EaC4H was related to these physiological indexes under salt stress condition too. The results showed the germination rate of transgenic tobacco plants of overexpressing EaC4H and EaC4H RNAi line was less than WT under the salt or drought stress. The results of chlorophyll content, MDA and POD content, SOD activity and CAT activity in transgenic tobacco had further proved that the WT tobacco have stronger resistance than the others. All these physiological results manifested that EaC4H gene is involved in reguLating the plant tolerance to salt or drought stress.