Preliminary Functional Analysis On LCD3 Gene In Rice

Cadmium(Cd)is a divalent heavy metal element,and excessive Cd accumulation can endanger human health and affect plant growth.By transcriptome sequencing analysis under Cd stress,it was found that rice OsLCD3 was induced by Cd.OsLCD3 has a phosphokinase domain,has no transport function,and is not a detoxification protein.It is of great theoretical significance to explore the mechanism and function of OsLCD3 response to Cd.In this study,the mutant lcd3 was obtained by CRISPR/Cas9 technology,and combined with transcriptome sequencing and yeast two-hybrid technique,the role of OsLCD3 in rice biological function and regulation of cadmium ion transport was studied.The main results are as follows:1.Constrcted OsLCD3-Cas9 knockout vector by using OsLCD3 genomic sequence to design gene editing target site.And obtained 43gene-edited knockout lines by genetic transformation in the background of Nipponbare.Then identified 23 positive plants,including 12 homozygous and 11 heterozygote.The CDS fragment of OsLCD3 gene was ligated to the pHB overexpression vector with 2×35S promoter by homologous recombination and transferred into rice.Four positive plants were identified.The lcd3-1(-6 bp),lcd3-19(-5 bp),OE-2,0E-3 mutant were selected for subsequent experiments.2.The main agronomic traits of lcd3-1,lcd3-19 and WT were observed and counted.It was found that the 1000-grain weight and seed setting rate of the mutant were significantly higher than that of WT.The seed of mutant was significantly larger than WT,but the seed germination rate was decreased significantly.3.The lcd3 mutant seeds appeared chalky,and the physicochemical properties of WT and mutant starch were analyzed.The total starch content of the mutant seeds was found to be significantly lower than that of WT,and the protein content was extremely significantly higher than that of WT.Observation and analysis of the over-expressed strains OE-2and OE-3 showed that the 1000-grain weight of over-expression mutant was significantly lower than that of WT,and the grain was significantly smaller than WT.4.Cd treatment of lcd3-1 and lcd3-19 knockout mutant seedlings showed that the growth state of lcd3-1 and lcd3-19 mutants was better than WT.The Cd content was measured,and the results showed that the Cd content of the underground portion and the underground portion of the mutant was significantly lower than that of the WT.5.The lcd3-1,lcd3-19 knockout mutant and WT were planted in soil with a total Cd content of 7.0 mg/kg,and the cadmium content in each organ of the plant was measured.It was found that the Cd content of the lcd3 mutant was significantly lower than that of WT in roots,stems,leaves,shells and grains.6.RT-PCR and qPCR analysis of tissue expression of OsLCD3.In the seedling stage,OsLCD3 is highly expressed in the aerial part,and the flowering stage is mainly expressed in stems,leaves,leaf sheaths,nodes and flowers.By GUS in vivo staining,it was mainly expressed on the leaves at the seedling stage,especially the leaf cushion had the highest expression.At the flowering stage,they were mainly expressed on the nodes,leaf cushion,Husk,anthers and stigmas.7.Screening rice cDNA library by yeast two-hybrid technique A total of 26 proteins that may interact with OsLCD3 were obtained.It is preliminarily determined by functional analysis that the interacting proteins obtained by screening may be involved in lipid transport andmetabolism;participate in intracellular transport and secretion,participate in certain signal transduction pathways in plants to cope with abiotic stress;may participate in ion transport;7 transmembrane proteins;in addition,there are kinases such as casein kinase,serine/threonine protein kinase,phospho-glycerate-kinase;it is speculated that the OsLCD3 gene may regulate other metal transporters to regulate cadmium transport in rice,and It may also play an important role in the growth and development of plants and the resistance to stress.8.Analysis of RNA-seq sequencing results showed that at the transcriptional level,the number of expressed genes shared by WT and mutant lcd3 was 37421,and the number of expressed genes unique to lcd3 was 2706.According to GO enrichment analysis,there are 52 genes with significant differences between WT and lcd3,of which 27 genes are up-regulated,25 genes are down-regulated,most genes are localized in cell membrane,and molecular function is related to binding.,regulate transcriptional activity,participate in a variety of biological processes such as plant signal transduction,positive response to stimuli,and cell growth.9.Subcellular localization analysis of OsLCD3.The endoplasmic reticulum red fluorescent marker Bip-RFP and pCUbi1390-OsLCD3-GFP were co-transformed into rice protoplasts.Under laser confocal microscopy,RFP fluorescence and GFP fluorescence could overlap well,indicating that OsLCD3 was localized on the endoplasmic reticulum of cells.In summary,OsLCD3 can not only regulate the absorption of Cd ions in rice,Deletion of the OsLCD3 gene reduces the accumulation of Cd ions in the rice,reduce the toxicity of Cd,but also regulate the grain size,affect the starch content and protein concentration in the grain,and participate in plant growth and metabolism.