| Frequent occurrence of extreme high temperature weather in recent years has seriously affected the growth and development of crops.When plants are subjected to high temperature stress,they can induce a large amount of HSP70 protein production.HSP70 family proteins as molecular chaperones can help plants resist the effects of high temperature stress.Micro RNA(mi RNA)is a type of 21-24 nt endogenous non-coding single-stranded small RNA which is widespread in eukaryotes.It regulates a variety of life activities in the plant by mediating the target gene cleavage or inhibiting its translation,such as participating in the process of plant heat stress.In this study,a new mi RNA,named novel-mi R311,was identified in the shoot tip of Brassica napus by high-throughput technology,and its target genes belong to HSP70 family.An overexpression vector of novel mi R311 was constructed and transformed into Arabidopsis and Brassica napus.The biological function of novel-mi R311 was clarified by phenotypic identification of transgenic plants after high temperature stress,as well as subsequent q PCR and soluble sugar content analysis.The main results are as follows.1.The novel mi R311 precursor sequence was compared with the Brassica napus and Arabidopsis genome databases,which found that it only existed in the first intron of the Bna A06g01660 D,no similar sequence was found in Arabidopsis.Then the Bna A06g01660 D was cloned in Brassica napus J572,and no novel mi R311 precursor sequence was found in Bna A06g01660 D sequence.The novel-mi R311 precursor and Bna A06g01660 D were cloned in Brassica rapa,Brassica oleracea,Brassica juncea and several varieties of Brassica napus.The results showed that the novel-mi R311 precursor could be cloned in all of the above materials,however,only the novel-mi R311 precursor which was cloned in rapeseed Zhongshuang 11 and Brassica rapa exists in Bna A06g01660 D,and the specific position in other materials remains to be studied.At the same time,its minimum folding free energy and secondary structure were analyzed,and it was found that the novel-mi R311 precursor could form a stable stem-loop structure.2.10 target genes of novel-mi R311 were identified by degradation group sequencing.It was found that 8 target genes belong to the heat shock protein HSP70 family,and the other 2 target genes belong to transduction protein encoded genes.The two target genes Bna Cnng03470 D and Bna A10g27060D(HSC70-1)were confirmed cleaved in Brassica napus by 5’-RACE technology.Sequence comparison analysis of 10 target genes with homologous genes in the Arabidopsis genome revealed that the target genes in rapeseed are very similar to the Arabidopsis homologous gene sequences.3.To further clarify whether novel-mi R311 achieves its function by mediating the cleavage of its target gene HSC70-1 in the plant abiotic stress response pathway.An overexpression vector of novel-mi R311 was constructed to transform Arabidopsis and rapeseed.4.The results of q PCR in Arabidopsis found that novel-mi R311 was expressed in transgenic Arabidopsis,but not in wild type,and its target gene expression level in Arabidopsis overexpression seedlings was significantly down-regulated.The q PCR results in Brassica napus showed that the overall expression level of novel-mi R311 in J572 was not high,but relatively high in roots and stems.5.The results of high temperature stress experiments showed that in Arabidopsis and Brassica napus,the growth potential and survival rate of novel-mi R311 transgenic positive seedlings were significantly lower than those of their corresponding control after high temperature stress.The q PCR results showed that the expression level of novel-mi R311 in rapeseed overexpression seedlings was significantly higher than that of control J572 before and after stress,and the expression level of its target gene HSC70-1 was significantly lower than that of control J572.The expression level of HSC70-1 increased as a whole after heat stress than before.After heat stress,the soluble sugar content in the overexpression seedlings seedlings of both Arabidopsis and rapeseed was significantly lower than that before heat stress,while in their corresponding control,the soluble sugar content was higher than that before heat stress. |
