Influence Of Nitrogen On Activity Of4-Coumarate:Coenzyme A Ligase(4CL) And Its Gene Expression In Eupatorium Odoratum

Eupatorium odoratum L. first appeared in Yunnan Province since1934, it has beenclassified as one of the100kinds of serious harmful invasive alien species in the world.“The evolutionary hypothesis of nitrogen allocation” hypothesis from Feng Yulong ismore representative in the study of control Eupatorium odoratum further spreadinvasion mechanism, the test uses native species-Mexico ecotype and invasivespecies-Xishuangbanna ecotype as materials, set three kinds of nitrogen level: highnitrogen (nitrogen content315.165mg/L), the normal nitrogen (nitrogen content210.11mg/L) and low nitrogen (nitrogen content105.055mg/L) to direct at therelationship between nitrogen and plant invasions.4-coumarate:coenzyme A ligase is the key enzyme in phenylpropanoid pathwayand lignin, flavonoids and other substances specific biosynthetic pathway. In this test,we discuss the relationship between Eupatorium odoratum phenylpropanoid metabolicpathway and nitrogen levels through4CL enzyme activity and gene expression. In orderto reveal the function of phenylpropanoid metabolic pathway in plant invasion so as toobtain the evidence of Eupatorium odoratum evolutionary response.This study has achieved the following:(1)4CL enzyme activity and related indicators under different levels of nitrogen:compared with the normal nitrogen levels, it has low4CL enzyme activity but highgrowth rate at low nitrogen levels, which may result in low lignin content, weakeningthe resistance to the external environment. Lower stem and leaf chlorophyll contentwould give rise to weaker photosynthetic capacity. Compared with the normal nitrogenlevels, it has higher4CL enzyme activity at high nitrogen levels which the stem abovethe leaves. It may be due to its low growth rate and stem lignin content higher thanleaves. While leaves have the higher chlorophyll content than the stem, suggesting thatmost of its nourishment supply leaf growth, nitrogen more allocation to thephotosynthetic apparatus, in order to reduce the allocation to the defense system, expandEupatorium odoratum invasion;(2) The comparison of Eupatorium odoratum from two ecotypes: Invasivespecies-Xishuangbanna ecotype growth better and quicker than the native species-Mexico ecotype. Xishuangbanna ecotype of4CL enzyme activity and4CLenzymatic specific activity are always higher than the Mexico ecotype under differentlevels of nitrogen;(3) Obtain part of the sequence in Eupatorium odoratum4CL gene: so farEupatorium odoratum4CL gene has not been cloned, this test combined withdegenerate primer design and RT-PCR method for encoding the conserved domain of4CL gene in Eupatorium odoratum, named for Eo4CL. The sequence length of426bp,encoding142amino acids. Sequence analysis showed that: Eo4CL protein containingthe4CL-like protein-specific catalytic active site GEICIRG. This gene encodes a proteinis similar to the other4CL protein;(4) Obtain part of the sequence in Eupatorium odoratumβ-Actin gene: Use thepublished Compositaeβ-Actin gene sequences in GenBank to design primers, PCRamplification to obtain reference geneβ-Actin sequence;(5) Influence of nitrogen on Eo4CLgene expression: To explore the expressiondifferences of Eo4CLgene from Xishuangbanna ecotype and Mexico ecotype, stems andleaves, different levels of nitrogen.