Isolation And Functional Analysis Of Cadmium-inducible Promoter In Rice

Rice is the main food crop in China. With the development of industry and agriculture, the heavy metals in environment and soil， especially cadmium accumulate in various tissues of rice, affecting the yield and quality of rice. Cadmium can not be degraded in the environment, can be a serious hazard to human health through the food chain. So reducing and eliminating cadmium pollution has become a serious problem. By altering the soil, optimization of water and fertilizer or flooding during the growing period may alleviate the toxic effect of cadmium on Rice, reducing the cadmium content in rice. But it will greatly increase the cost of cultivation. Therefore, genetically modifing rice varieties resistant to Cd and breeding varieties with low cadmium accumulation is an economical and effective way to reduce the cadmium contamination of rice.The main stress response of plant is the transcriptional regulation of gene expression, depending on the interaction between the promoter and the transcription factor. Inducible promoters are expressed only under certain circumstances,avoiding plant injury caused by excessive expression of target genes. Therefore, inducible promoters provides an important resource for the study of the regulation of stress tolerance mechanism. Moreover, the current study focused on the function of genes, and little research on rice cadmium inducible promoter. This paper we identified and isolated three promoters responsible to cadmium, and analyzed their activity. The results are as follows:(1) By microarray analysis and qRT-PCR identification, we find out three main cadmium induced genes, OsGSTU37、OsHSP18.6 and OsGSTU5 respectively.(2) Cloning of the above three rice cadmium responsive gene promoters, named POsGSTU37、POsHSP18.6 and POsGSTU5 respectively. We construct expression vectors of the fusion of these three promoter and GUSplus reporter gene. By Agrobacterium mediated genetic transformation we obtained 20,25 and 27 strains of positive transgenic rice plants.(3) Of the three POsGSTU37、POsHSP18.6 and POsGATU5 promoters by qualitative and quantitative analysis of expression activity of Cd induced tissue. GUS staining of the corresponding promoter T3 transgenic plants root, stem and leaf tissues, compared with untreated, its root, stem and leaf tissues have different degrees of color after inducement by cadmium, indicating that these three promoters are cadmium induced promoters. At the same time, the three promoters POsGSTU37,POsHSP18.6 and POsGATU5 activity of qRT-PCR quantitative analysis showed that by cadium inducement for 24h the activity of POsGATU5 and POsGSTU37 is fair to POsACTIN, a constitutive promoter, POsHSP18.6 showed higher activity than POsACTIN induced by cadmium, the aerial parts induced by cadmium were 1.33 times than POsACTIN, the root induced by were 5.38 times than POsACTIN.(4) In the short term of cadium induction(72h), promoters POsDSTU37,POsHSP18.6 and POsGSTU5 exhibit different patterns of cadmium-induced responses.With the extension of cadmium induction time, POsGSTU37 activity increased, reaching a maximum at the time of 72h,880 times than which is not induced. The activity of POsHSP18.6 was strongly upregulated in the early period of cadmium induction, reaching highest at 8h, about 1220.8 times, but the activity decreased with the extension of time. POsGSTU5 has high basal expression, reaching 24 times at 4h. Then cadmium-inducing activity is maintained at a relatively stable level.(5) Quantitative analysis of the three cadmium inducible promoters expression patterns in the long cadmium-induced shows POsGSTU5 and POsGSTU37 root activity was significantly higher than the leaves. This may be due to different concentrations of Cd accumulation in different tissues of rice. GUSplus driven by POsGSTU37,expression levels of cadmium with prolonged stress time increasing the highest in 20 days. The difference is, expression level GUSplus of in POsGSTU5 and POsHSP18.6 gene transfer plants does not depend on the length of cadmium stress time. Compared with the GUSplus expression in roots and leaves of transgenic plant without Cd for four days, the highest multiples POsGSTU37 roots for 20 days and POsHSP18.6 leaves for 5 days.(6) We analysed induction activity of different metals Ni、Pb、As、Co、Cu、Fe、 Zn and Mn on cadmium-induced promoter transgene plants. Under Ni induction, GUSplus induction activity in POsGSTU5 gene transgenic plants was strongest, which induces multiple times up to 119.5. Under Pb and As induction, POsGSTU5 activity has also been strongly upregulated. Under metals Cu、Fe、Zn、Mn and Co, induction expression is very low. Metals Ni、Cu、Pb、As and Co can strongly induce POsGSTU37, but metal Mn induced lowly, but also increased the expression of GUSplus(P<0.01), however, effect was not significant on POsGSTUu37 activity raised by Zn and Fe. Compared with the negative control, induction of various metals increased GUSplus expression to a large extent in POsHSP18.6 transgene plants, where in, Ni、Cu、As and Co is much stronger than Pb、Fe、Mn and Zn on POsHSP18.6 induction ratio.(7) We made functional analysis of the three cadmium inducible promoters inspectively. Currently we only cloned the corresponding truncated promoter with a total length of cadmium inducible promoter as a template, and constructed fusion expression vector of promoter and GUSplus report gene, POsGSTU5 cadmium response element was located in the-2240～-1572bp, POsGSTU37 was induced by the truncated promoter, and the response element of POsHSP18.6 was located in the -437～-190bp.The paper found and identified the three cadmium inducible promoters, carried out qualitative and quantitative analysis of these three cadmium inducible promoter inspectively. The three cadmium-inducible promoters provides an important resource in the study of rice cadmium stress regulation mechanism. And they provides an effective method in cultivation of resistant varieties of cadmium and cadmium low absorption. Next step we prepare to peform functional analysis of cadmium inducible promoter. Analysis of the smallest key element of the promoter of cadmium-induced and explore its mechanism of actionis help to understand the mechanism of cadmium tolerance in rice.