Analysis And Validation Of MiRNA Related To Cadmium Stress In N.tabacum And N.rustica

Tobacco is an important economic crops in my country.Cadmium(Cd)is a toxic heavy metal that is easily absorbed and accumulated by tobacco,affects the growth and development of tobacco,and can enter the human body through smoke to threaten human health.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs that play an important role in biological processes such as plant growth and development,cell proliferation,stress response,and hormone regulation.With the development of high-throughput sequencing,more and more abiotic stress-related miRNAs and their target genes have been accurately identified,but there are relatively few studies on cadmium stress.In this paper,Nicotiana safflower K326(cadmium accumulation in leaves)and Nicotiana tabacum Maxopka(cadmium accumulation in roots)were used as experimental materials,and high-throughput sequencing technology was used to screen and identify miRNAs related to cadmium stress,and then construct overexpression transgenic plants to verify miRNAs Role in cadmium tolerance in tobacco.This study will help to reveal how different genotypes of tobacco genotypes regulate miRNAs to adapt to cadmium stress at the post-transcriptional level,and provide support for the discovery of stress resistance genes and genetic improvement in plants.The main findings are as follows:(1)miRNA screening and identification.Four small RNA(Small RNA,s RNA)libraries of two genotypes K326 and Maxopka treated with cadmium in tobacco and control were constructed.Through miRNA high-throughput sequencing and analysis,there were121 differential miRNAs in the two genotypes before cadmium treatment,including 58 known miRNAs and 63 novel miRNAs;a total of 126 miRNAs were significantly different between the two genotypes under cadmium treatment,including 60 known miRNAs and 66 new miRNAs.Target gene enrichment analysis showed that differential miRNAs were mainly involved in five aspects related to cadmium tolerance in cell physiology,namely miRNAs related to heavy metal transport(miR159,miR167a),miRNAs related to sulfur metabolism(miR395 a,miR156f),and miRNAs related to oxidation.Reduction reaction-related miRNAs(miR398,miR408),miRNAs involved in regulating cell growth(miR319 a,miR6145 a,miR156 f,miR6161 a,miR6161c)and ion homeostasis-related miRNAs(miR6149 a,miR482b-3p).(2)Construction of overexpression vector and genetic transformation.Among the 126 differentially expressed miRNAs,the expression of miR395 a involved in sulfur assimilation was significantly different between Maxopka Cd treatment and control,but there was no significant difference between K326 Cd treatment and control;miR319 a involved in regulating root cell growth was significantly different between K326 Cd treatment and control.There were significant differences in expression between controls,but there was no significant difference in expression between Maxopka cadmium treatment and controls.miR395 a and miR319 a were selected to construct the miR395 a overexpression vector of K326 and the miR319 a overexpression vector of Maxopka,respectively,and carried out Agrobacterium-mediated genetic transformation.miR395 a and miR319 a overexpression plants were obtained,and 18 miR395 a overexpression positive seedlings and 12 miR319 a overexpression positive seedlings were identified by hygromycin gene identification.(3)Validation of miR395 a overexpression plants of K326.After one week of treatment with 10 μmol/L Cd Cl2 in wild-type and over-expression plants,the cadmium accumulation,glutathione(GSH)content and glutathione reductase(GR)activity of over-expression plants were significantly higher than those of wild-type K326.Among them,the accumulation of cadmium in roots of miR395 a overexpressing plants was 2.38 times that of wild type,the cadmium content in leaves was 1.17 times that of wild type,and the cadmium transport coefficient(TF leaf/root)was 0.5 times that of wild type.The GR activity was 1.82 times and 1.76 times that of the wild type,respectively.The expression level of wild-type K326 miR395 target gene SULTR2;1 was significantly higher than that of transgenic K326.The results indicated that tobacco could accumulate more cadmium in roots through the interaction of high expression of miR395 a and its target genes.(4)Validation of Maxopka miR319 a overexpression plants.After the wild-type and over-expression plants were treated with 10 μmol/L Cd Cl2 for one week,the cadmium accumulation,cadmium uptake rate and root-related indexes of the over-expression plants were significantly lower than those of the wild-type Maxopka.Among them,the accumulation of cadmium in roots of miR319 a overexpressing plants was 0.62 times that of wild type,the accumulation of cadmium in leaves was 0.71 times that of wild type,and the cadmium transport coefficient(TF leaf/root)was 1.2 times that of wild type;The absorption rates of cadmium at 0~6h and 8~48h were 0.51 and 0.61 times that of the wild type,respectively.The results indicated that tobacco could inhibit the rate of cadmium uptake and accumulate more cadmium in leaves by overexpressing miR395 a.