Construction Of CDNA Library And Studying Of Phenylalanine Ammonia-Lyase (PAL) Gene Family In Eupatorium Adenophorum

Eupatorium adnophorum Spreng. is one of the most disastrous weeds in China. It originated from Mexico and Costarica. Now it can be found in more than 30 countries and regions. Eupatorium adenophorum entered into our country via the border of Sino-Burma since 1940s and spread extensively in south and southwestern regions of China. It spread fast and has drawn the attention of the society. A lot of research shows that E. adenophorum has strong allelopathy, and it can produce the allelopathic substances which can inhibit the growth of surrounding plants. 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one (HHO) is one of the most important allelopathic substances.Our research is focused on secondary metabolic pathway and molecular regulation of HHO through cloning and function analysis of HHO metabolism related genes. It can provide theoretical basis for comprehensive control of Eupatorium Adenophorum.A cDNA library of Eupatorium adenophorum was constructed by using SMART system in the first time. According to the evaluation on quality, the library had a high titer of 1.23×107 cfu/mL, the average insert of fragments size was about 900 bp and 98% recombinant percentage, which indicated that the library had high quality for cloning target genes. The cDNA fragment of Ribulose diphosphatecarboxylase (RuBP) was obtained by screening this cDNA library(GenBank ID: FJ752421), also a feasible method was established for screening target genes.We cloned two genes from Eupatorium adnophorum Spreng.. In this study, phenylalanine ammonia-lyase(PAL) (GenBank ID: GQ259802), cinnamate 4-hydroxylase(CA4H) (GenBank ID: GQ259803). According to the real-time quantitative PCR result, we found that this two genes are related with the metabolism of allelochemical (HHO). We also cloned two HHO-related genes from Arabidopsis thaliana: 4-Coumarate : coenzyme A ligase(4CL), caffeic acid O-methyltransferase(COMT). According to the result of semi-quantity RT-PCR, we found that 4CL and COMT were related with the metabolism of HHO Analogues.Among these four genes, the change of expression of PAL was the most significant. Under the high or low dose inducing of HHO, the expression level of PAL gene increased significantly.Southern blotting shows that PAL gene family has three members in E. adenophorum. The PAL gene was cloned by Rapid amplification of cDNA ends(RACE) method. We obtain the full-length sequence message of EaPAL1 and EaPAL2 (GenBank ID: GQ259804, GQ259805). According to the result of real-time quantitative PCR by using gene specific primers, We found that the expression level of EaPAL2 gene increase more significantly than EaPAL1 under high or low dose inducing of HHO. This means that EaPAL2 is closely relationship with HHO metabolism.Furthermore, We constructed RNA interference vector of PAL conserved region, which will be transformed into Eupatorium adenophorum. It is helpful to investigate the function of this gene.