In The Process Of Germination Of Expression Of ICL Gene In Zanthoxylum Dissitum Seeds Treated With GA₃

Zanthoxylum dissitum Hemsl is one of the important valuable and rare medicinal plant in China,all its roots,stems and leaves,which could activate blood circulation to dissipate blood stasis,resolve toxin and disperse swelling,promote union of fracture healing,are important main raw materials for many Chinese patent drug for sprain,lumbago ect.In recent years,because of domestic production scale,and Zanthoxylum dissitum Hemsl seed natural germination rate is low,and the growth speed is slow,resulting in Zanthoxylum dissitum Hemsl natural resources is poor,can not meet the demand of the related pharmaceutical factory.Zanthoxylum dissitum Hemsl seeds have the high oil content.In the early stages of germination,Zanthoxylum dissitum Hemsl mainly use the fat in which seeds stored as the main energy material,and through the glyoxylic acid recycling pathway to convert fat material into carbon source.In this study,we studied the expression of isocitrate lyase gene during the process of seed germination of Zanthoxylum dissitum Hemsl from the molecular level by using gene cloning and qRT-PCR technology in the process of gibberellin-treated germination which revealed the regulation of seed oil mobilization at the early stage of its germination about Zanthoxylum dissitum Hemsl.It also provided some reference for solving the problem of low seed germination rate.The following progresses have been made in the research work:(1)The expression levels of six potential reference genes(GAPDH,ACT,UBQ5,CYP,TUA,18SrRNA)were investigated in the GA and the water treated Zanthoxylum dissitum Hemsl seed by SYBR green real-time RT-PCR under different germination period.Then use NormFinder,Bestkeeper and geNorm Software to filter the most stable expression gene.Finally,ACT and UBQ5 with the most stable expression should be screened out as reference gene in all samples of Zanthoxylum dissitum Hemsl seed.(2)We treated the seeds of Zanthoxylum dissitum Hemsl with GA and water respectively,and detected the expression of ZdICL gene at different germination stages by qRT-PCR.The results showed that the expression level of ZdICL was lower than that of the seeds treated with GA on the first day of germination.It may because the oil metabolism of seeds was inhibited in the early stage of seed germination,so the H2O2 were reduced,which was produced by β-oxidation during the process of fatty acid mobilization,and the oxidative damage of the free radicals during the bulging process were avoided.The expression of ZdICL increased significantly from the second day of germination,which was due to GA promoted the metabolism of various antioxidant enzymes by gradually degrading ABA,and the antioxidant capacity of various antioxidants in seeds increased gradually.At the same time,its ability of inhibit the metabolism of fat mobilization was abating,and the metabolism of oil in the seeds was more active,so,it provided carbon source and energy for the seed and promoted the seed’s germination.The results indicated that GA could inhibit the mobilization of oil and fat in the early stage of germination of shells,reduce the H2O2 produced by β-oxidation in fatty acids,avoid the damage caused by seeds and protect the normal germination of the pepper seeds.(3)The CDS full length sequence of ZdICL gene was cloned by RT-PCR strategy with the template of RNA extracted from Zanthoxylum dissitum Hemsl seeds.The full-length of ZdICL CDS was 1728bp,encoding 575 amino acids.The molecular weight and theroretical isoelectric point(p1)of the deduced ZdICL protein were 64460 and 6.94,respectively.The protein of ZdICL was stable,hydrophobic and soluble.It revealed that ZdICL had closer relationship with ICLs from Citrus sinensis than from other plants by phylogenetic tree analysis.Secondary and tertiary structures indicate that ZdICL gene contained TIM_phosphate_binding superfamily conserved domain and a hexapeptide conserved domain with amino acid sequence KKCGHM.(4)A CDS sequence of ZdICL gene which contain Bam HI and Xho I restriction enzyme sites in its 5’ and 3’ terminal was achieved by PCR in the recombinant plasmid as template.The CDS sequence through this two restriction enzyme sites digested and inserted to pET-28a(+)vector,a prokaryotic expression vector of pET-28a-ZdICL was constructed.This recombinant expression vector was transformed to host bacteria E.coli BL21(DE3),then after,IPTG was added to induce expression.By exploring the temperature,time points as expression conditions,we found that the optimal expression condition for getting fusion protein pET-28a-ZdICL was 20 ℃,19 h and 1 mM IPTG.The target protein was purified by Ni-IDA column affinity chromatography.The concentration of the target protein was 610 μg/ml and the recombinant ICL was purified in highly active state with a specific activity of about 362.84 nmol/min/mg prot.