Cloning And Expression Analysis Of Hevea Brasiliensis MVK Gene Promoter

Rubber tree(Hevea brasiliensis),a rubber-production crop,has the largest planting scale in the world.As the most important source of natural rubber,rubber tree has been a research focus in many countries.Its product(natural rubber)is an extremely important industrial raw material and many developed countries are heavily dependent on imports.Therefore,natural rubber is also an important strategic material.In order to elucidate the mechanism of rubber biosynthetic regulation in rubber trees and lay a foundation for molecular breeding of high-yield rubber trees,the promoter sequence of the gene encoding the mevalonate kinase(MVK),the first ATP-dependent acid kinase in the mevalonate pathway,was cloned and the bioinformatics analysis and transgenic technology were used to study its regulation mechanism.The research results are as follows.According to the results of rubber genome sequencing,a 1696bp rubber MVK gene promoter sequence was cloned and submitted to the promoter online analysis sites for cis-acting element prediction.The results of prediction showed that the promoter contains up to 71.34%AT,CAAT-box and TATA-box(which are typical core components of the plant promoter),TCA-element and TGACG-motif(hormone response elements),CAT-box(tissue-specific expression elements)and Sp1(photoreactive elements),etc.At the same time,other gene promoter sequences in the rubber synthesis pathway were analyzed,and the TCA element(associated with salicylic acid regulation)and the CGTCA-motif(related to jasmonic acid regulation)have been found to be widely distributed in these promoter sequences.The cloned HbMVK full-length promoter(named M5)was used as a template and a series of upstream primers was designed based on the predicted position of the regulatory elements.A series of 5'-deleted promoter sequences were cloned and the length were 325 bp.725 bp,1221 bp and 1386 bp respectively(sequentially named M1,M2,M3,and M4).Five promoter sequences(including the full length)were connected to the upstream of the GUS gene and transferred into Arabidopsis plants by Agrobacterium mediated flower-dipping method.Then,stably expressed transgenic lines were selected and the expression characteristics of GUS gene were detected by histochemical staining and fluorescence analysis.The detection of GUS activity in transgenic Arabidopsis plants showed that the Ml promoter had no activity in transgenic Arabidopsis,probably because of the lost of important core elements.The M2 promoter activity was the highest among the five promoter fragments(just next to that of the CaMV 35S promoter),while the M3,M4,and M5 activities were sequentially decreased,possibly due to the existence of a complex interaction of the upstream of 5' end of the HbMVK gene promoter with the trans-acting factors in the heterogeneous expression system.For the GUS staining of transgenic Arabidopsis plants,the full-length HbMVK promoter(M5)was observed to have very weak expression in hypocotyls of transgenicArabidopsis seedlings at 5 days and no expression in other parts and other seedlings beyond 5 days;The M4 promoter showed weaker expression activity in the hypocotyls;the M3 promoter was relatively active in the cotyledons and stems;the M2 promoter had strong activity of driving the GUS gene expression in all parts of the Arabidopsis above the ground.The Ml promoter was essentially inactive under heterogeneous expression.This showed that the HbMVK gene promoter can drive the GUS expression in a tissue-specific and time-specific manner in Arabidopsis seedlings but its specificities are related to its sequence length.In order to study the effects of exogenous hormones and environmental stresses on HbMVK gene promoter activity,the transgenic Arabidopsis plants of M5(full-length promoter sequence)and M4(1386 bp 5'-deleted promoter sequence)were treated with ethephon,jasmonic acid,auxin,cytokinin,salicylic acid,gibberellin,cold and short photoperiod.The changes of GUS activity after treatment were largely verified the function of the predicted regulatory elements located in different regions of HbMVK gene promoter.The study demonstrated that ethephon,gibberellin,and cold stress down-regulated the activity of M4 and M5 promoters,while the effects of auxin and salicylic acid were opposite;Cytokinin has no significant effect on promoter activity.Comparing the activity of the full-length promoter and 1386 bp 5'-deleted promoter sequence under various treatments,the activity of the full-length promoter of HbMVK gene was found to be weaker than that of M4 under almost all treatments.The reason is possibly that the 300 bp about upstream region of the 5'-end of M5 promoter had an inhibiting effect on GUS activity by a negative interaction of the region with the trans-acting factors in the heterogeneous expression system.