Preliminary Study On The Molecular Mechanism Of Bnvit-l2 Gene Involved In Lateral Root Development And Bioaugmentation Of Rapeseed Under Iron Stress

Rapeseed is a widely planted oil crop in China.In addition to oil,it can also be used for vegetable,medicinal,flowering and fertilizer.However,during the growth and development of rapeseed,many factors affect its yield,such as biological stress such as pathogens and abiotic stress such as heavy metal ions.Heavy metal stress has a strong destructive,so it is important to maintain metal homeostasis in plants,and metal transporters play an important role in this process.In this paper,the vacuolar iron transporter gene Bn VIT-L2 in Brassica napus L.was studied and its iron tolerance function was preliminarily analyzed.The main research results are as follows:1.The vector p K7FWG2.0-Bn VIT-L2-e GFP was introduced into rape K407 by hypocotyl tissue culture.The transformed plants were identified by PCR and the expression levels were analyzed to obtain three positive transformed lines,which provided materials for subsequent experimental analysis.2.To investigate the iron tolerance of Bn VIT-L2 in rape,Bna VIT-L2-OE and K407were grown under different concentrations of iron stress.It was found that compared with K407,the number of lateral roots and leaf area of Bna VIT-L2-OE were significantly increased to measure plant growth.It indicated that the plants overexpressing Bna VIT-L2-OE had higher iron tolerance,and Bn VIT-L2 gene was involved in the lateral root development of rape under iron stress.And the iron content in Bna VIT-L2-OE was higher,suggesting that Bna VIT-L2 gene has the function of storing iron ions and can play a role in biological enhancement.3.In order to further explore the molecular mechanism of Bna VIT-L2 iron tolerance,Bna VIT-L2-OE and K407 were treated under iron stress,and Ribo-seq was used to detect the differences in translation levels of different rapeseed varieties.Data analysis showed that differential genes were enriched in signaling pathways such as divalent metal ion transport,carbon metabolism,phenylpropanoid metabolism,hormones,and antioxidant.This is similar to our previous Arabidopsis transcriptome data,suggesting that Bna VIT-L2 plays a role in transporting metal ions,and that carbon metabolism,ROS and other signaling pathways may also be involved in the regulation of lateral root development by Bna VIT-L2 under iron stress.4.In order to further detect whether ROS signaling pathway is related to Bna VIT-L2gene regulate the growth and development of rape under iron stress,DAB and NBT staining were performed on Bna VIT-L2-OE and K407 rape grown under iron stress,and the levels of O₂^·-and H₂O₂ were detected.The results showed that compared with K407,the staining degree of Bna VIT-L2-OE was shallow and the amount of accumulated reactive oxygen species was small.Differential gene enrichment was found in the reactive oxygen species pathway in both transcription and translation groups.It was speculated that Bna VIT-L2-OE had higher tolerance to iron stress than K407.ROS accumulation decreased,thereby reducing oxidative damage and promoting plant growth.ROS mechanism is involved in the growth and development of Bna VIT-L2under iron stress.5.The environment and plant genotypes affect rhizosphere microorganisms.In this chapter,16S sequencing analysis was used to study microbial diversity.Compared with the control,the relative abundance of nitrogen-fixing bacteria in rhizosphere microorganisms of the soil growing Bna VIT-L2-OE increased significantly,and the iron tolerance of Bna VIT-L2-OE was related to biological nitrogen fixation.Under iron stress,the composition of rhizosphere microbial species in the soil growing Bna VIT-L2-OE was closer to that under iron-free stress than that in the control.This may be due to the fact that Bna VIT-L2 could transport iron to vacuoles and enhance the iron tolerance of Bna VIT-L2-OE,resulting in less changes in root exudates and small differences in rhizosphere microbial species composition.6.Bn VIT-L2-Cas9 and Bn VIT-L2-RNAi vectors were also constructed and transformed into B.napus.Seven Bna VIT-L2-RNAi positive transformation lines and three CRISPR/Cas9-Bna VIT-L2 were preliminarily identified,respectively,which provided materials for subsequent experimental analysis,and their functions need to be further verified.