| Yam(Dioscorea opposita Thunb.)is a medicinal and food homology plant with tuber as the main product organ.The size and quality of tuber directly affect the yield and economic value of yam.With the improvement of people's living standard,the demand for yam yield and quality increases year by year.Starch,as the main component of yam tuber,is one of the important indexes of yam yield and quality breeding.Starch accumulation and degradation affect the growth and development of tuber,and its content directly affects the quality of yam tuber.Therefore,it is of great significance to excavate the related to development and starch accumulation to improve the quality of yam.This study measured tuber length,circumference,dry weight and fresh weight morphological indexes at 105,120,135,150 and 165 days after planting,using Bikeqi(B1)and Dahechangyu(DHCY)as experimental materials.Bioinformatics analysis of B1 transcriptome sequencing data was performed to screen differentially expressed genes related to tuber development and starch accumulation,and their accuracy was verified by q PCR.The cDNA length of yam DoVHAb2 gene and DoClpB3 gene were cloned by RACE technology,and the ORF of these two genes were analyzed by bioinformatics;The g DNAs were cloned and their gene structures were analyzed.Transient expression vector was constructed and injected into tobacco leaves to observe the subcellular localization of genes.RT-q PCR was used to analyze the gene expression in different growth and development stages of yam tuber,and the relative expression levels in different organs-tuber,stem and leaf were analyzed in the period of the highest relative expression level of tuber.During the growth and development of yam,the correlation analysis of starch related physiological indexes and relative gene expression in tubers was carried out.Plant expression vector of gene was constructed and transformed into tobacco by Agrobacterium-mediated method to identify the function of transgenic tobacco.The main research results are as follows:1.The change trend of starch content in B1 and DHCY was basically similar,both of which increased first and then decreased.The content of sucrose decreased.At the time of tuber harvesting,starch content in B1 and DHCY accounted for about 80%of its dry weight.There was a significant negative correlation between ATPase activity and starch content.There were significant negative correlations between chlorophyll content and sucrose content,sucrose content and starch content.The tuber dry weight,starch content,sucrose content and ATPase activities of B1 were all higher than those of DHCY at each stage of tuber development.The results indicated that ATPase directly regulates starch synthesis in tubers,chlorophyll content affected starch synthesis by affecting sucrose content and B1 was superior to DHCY in dry matter and starch content.2.The transcriptome of 15 RNA samples from B1 was sequenced and 171 554 unigenes were obtained from the sequenced original reads by bioinformatics method.17 218 Unigenes were annotated by gene GO function,which were divided into three categories: there were 7 516 unigenes in biological processes,5 748 unigenes in cell components and 3 954 unigenes in molecular functions.KEGG pathway analysis showed that the pathways related to tuber enlargement mainly included metabolic,plant hormone signal transduction,plant-pathogen interaction,endoplasmic reticulum and endocytosis pathways.Through transcriptome sequencing analysis of five growth and development stages of B1,147 co-expressed DEGs were screened,which were the key DEGs for the growth and development of yam.The q PCR verification showed that the q PCR results in the tuber of yam were 85% consistent with the transcriptome data,indicating that the DEGs data in the growth and development period of yam was highly reliable and accurate.3.The full-length cDNA of DoVHAb2 was cloned from B1,and the Gen Bank registration number was MK858181.The full-length cDNA was 1 926 bp,the ORF was 1 467 bp,encoding 488 amino acids.The 5' non-coding region length was 84 bp,and the 3' non-coding region length was 298 bp.The closest evolutionary relative to the palm.The structure of DoVHAb2 gene was divided into 14 exons and 15 introns.The protein was localized in the cytoplasm.In both cultivars,the expression of DoVHAb2 gene was the highest in tubers and the lowest in leaves.And the relative expression level of DoVHAb2 gene positively regulates the starch synthesis in yam tuber.These results indicate that DoVHAb2 gene is specifically expressed in tubers and regulates starch synthesis in tubers,thus controlling the growth and development of yam.4.DoVHAb2 was successfully transferred into tobacco genomicDNA,with a conversion rate of 92.3 %,and was also expressed at the transcription level.The starch content,sucrose content,AGPase,SSS and ATPase activities in transgenic tobacco were significantly higher than those in wild-type tobacco.The differentiation ability of the transgenic DoVHAb2 gene was significantly higher than that of the wild-type tobacco leaves under salt stress.Under salt and drought stress,the expression of DoVHAb2 gene in transgenic tobacco was significantly higher than that in non-stressed plants.The results showed that the expression of DoVHAb2 gene in transgenic tobacco was up-regulated under stress,and the salt tolerance and drought resistance of transgenic tobacco were enhanced.5.The full-length cDNA of DoClpB3 was cloned from B1,and the Gen Bank accession number was MN636784.The full length of the cDNA was 2 583 bp,the ORF was 2 055 bp,and it encoded 684 amino acids.The length of the 5' non-coding region was 99 bp,and the length of the 3' non-coding region was 428 bp.The evolutionary relationship with jujube is relatively close.DoClpB3 gene had 9 exons and 8 introns.DoClpB3 was localized in chloroplasts.The expression of DoClpB3 in two varieties of yam is the highest in tuber,and the lowest in leaf.The relative expression of DoClpB3 gene was negatively correlated with the starch content in yam tuber.It was positively correlated with sucrose content and chlorophyll content.These results indicated that DoClpB3 gene may directly affect the content of chlorophyll in yam and determine the content of sucrose,thus indirectly affecting the content of starch in tubers.6.DoClpB3 was successfully transferred into tobacco genomicDNA with a conversion rate of 91.7% and expressed at the transcription level.It was found that the upper epidermis,lower epidermis and palisade tissues of transgenic tobacco were thicker than those of wild-type tobacco,and the total leaf thickness was increased by42.2% compared with that of wild-type tobacco.The chloroplast volume of transgenic tobacco was larger than that of wild-type tobacco,the number and size of starch grains were also higher than that of wild-type tobacco,and the number of osmiophilic grains was less.The content of chlorophyll and starch in DoClpB3 transgenic tobacco was significantly higher than that in wild-type tobacco.These results indicated that DoClpB3 affected starch content by affecting leaf structure and chloroplast size and structure. |
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