Screening,Identification And Expression Vector Construction Of Long Non-Coding RNA Related To Cadmium Stress In Rice Roots

The problem of heavy metal pollution in soil is becoming more and more serious,which has aroused widespread attention from all walks of life.Heavy metals not only affect the normal growth and development of many plants,but also are brought into the food chain,seriously threatening human health.Cadmium（Cd）is one of the most toxic heavy metals,and the incidents of Cd exceeding the standard of Cd in agricultural products is common,which has aroused widespreas concern of the society.Rice（Oryza sativa L.）has been one of the crops that people often eat since ancient times,Cd pollution not only affects the growth and development of rice,but also affects its yield and quality,and even seriously affects human health.Long non-coding RNA（lncRNA）is a kind of non-coding RNA with a length more than 200 nt.Previous studies have shown that lncRNA is involved in many improtant regulatory processes,such as X chromosome silencing,genome imprinting,chromatin modification,transcription activation,transcription interference and nuclear transport.However,there are still few studies on the molecular mechanism of lncRNA in response to Cd stress in rice.Therefore,this project used the genome-sequenced rice varietie Nipponbare as the material,and the final concentrations of Cd Cl₂were 0μM and 25μM by the way of solution culture in the stress test.Using rice roots treated with Cd for 2 days as materials,RNA-seq sequencing was carried out to screen several lncRNAs that play an important role in the process of rice response to Cd stress,so as to provides a theoretical basis for comprehensively elucidating the molecular mechanism of rice response to Cd stress,and provides a new idea for scientific guidiance of rice production.The specific experimental results were as follows:（1）1380 lncRNA sequences were obtained by RNA-seq sequencing data analysis,including 785 lncRNAs of intergenic type（u）,219 lncRNAs of antisense type（x）,and 211 lncRNAs of intron type（i）,124 lncRNAs of bidirectional type（j）,and 41 lncRNAs of sense type（o）.In the structural analysis of lncRNA and m RNA obtained by sequencing,more than 77%of the total m RNAs and 72%of the lncRNAs were over 1000bp and 300bp respectively.There were differences in the number of structural exons between m RNA and lncRNAs.The proportion of m RNA with different number of exons was evenly distributed,while lncRNAs was mainly composed of one or two exons,lncRNAs with only one exon accounted for 60%.（2）Among the 1380 lncRNA screened,51 lncRNAs were found to be differentially expressed under Cd stress,of which 35 lncRNAs were induced by Cd and 16 lncRNAs were inhibited by Cd.Further functional analysis of GO and KEGG revealed that they were mainly involved in cell wall formation,peroxide clearance,GSH metabolism,GST activity,ion transport,secondary metabolism and stress response,and were highly enriched in GSH metabolism,ABC transporter,plant pathogen interaction and phenylpropionic acid synthesis.At the same time,the expression levels of some differential lncRNAs were verified by q RT-PCR,and the expression trends were consistent with the sequencing results.Subsequently,we further analyzed the expression characteristics of these differential genes in different parts of rice in response to 0μM,25μM,and 50μMCd treatment stresses,and found that the expression of some lncRNAs is tissue-specific.The focus was on research object of a newly discovered Cd induced o-type lncRNA-4899,and the lncRNA was named GSLNCR1（GST Sense long non-coding RNA）.Bioinformatics analysis showed that nine of the 10 CIS regulated target genes of GSLNCR1 were Os GSTs（LOC＿Os10g38470、LOC＿Os10g38489、LOC＿Os10g38610、LOC＿Os10g38580、LOC＿Os10g38590.1、LOC＿Os10g38600、LOC＿Os10g38630、LOC＿Os10g38590.2和LOC＿Os10g38540）,and one was the precursor gene of receptor like protein kinase2（LOC＿Os10g38450）involved in plant pathogen interaction.The nine GSTs all had two typical conserved domains（GST-N-Tau and GST-C-Tau）of the Tau subfamily of GST family.During this period,the full length of GSLNCR1 gene was cloned,and the overexpression and interference expression vectors of GSLNCR1 were constructed by using 1300UR-s GFP overexpression vector and p FGC1008 interference expression vector respectively,which laid a foundation for the further study of the biological function of GSLNCR1 in response to Cd stress in rice.