Identification And Functional Research Of Genes And MicroRNA Related To High-Efficiency Transport Of Photosynthate In The Siliques Of Brassica Napus L.

Rapeseed（B.napus L.）is one of the three major oil crop in the world,and also the oil crop with the largest planting area in our country.But unfortunately,as an economic crop that mainly harvests seeds,the harvest index（HI）of cultivars is only 20-30% approximately compared to the plants of rice and peanuts which own more than 50% HI.Therefore,increasing the harvest index of rapeseed is particularly important for increasing the yield of rapeseed.Among the three factors of “source,flow,and sink” that affect the harvest index,rapeseed has a sufficient source and a larger sink,so studying of flow which reflects the transportation and distribution of photosynthate has become a breakthrough to improve the harvest index of rapeseed.The photosynthesis of rapeseed in the green-pod stage can provide more than 50% photosynthetic product to the grain yield,the activity of Sucrose Phosphate Synthase in pericarp affects the conversion of starch and sugar,and then changes the intensity of carbohydrate transfer from pericarp to seeds,further affects the trait of seed weight per silique index（SPSI）and ultimately forms the traits of high HI.Our study determined the trait of SPSI and thousand-seed weight（TSW）as reference traits reflecting the transport efficiency of carbohydrate in siliques.We perform the genome-wide association study（GWAS）with the trait of SPSI and TSW among the 588 materials in resequencing population in three consecutive years,combined with the transcriptome and small RNA sequencing analysis of materials with extremely high and low SPSI as well as TSW,finally 12 candidate genes and 9 miRNAs that may be related to the transport efficiency of photosynthetic products in rapeseed silique were screened.The function of sucrose phosphate synthase2F（BnaSPSA2-2）and bna-miR168 a were identified by transgenic methods,it was found that the heterologous overexpression of BnaSPSA2-2 in Arabidopsis resulted in the phenotype of faster vegetative growth and early flowering,eventually form high-SPSI and high-HI plants.After overexpressing and interference-expression of bna-miR168 a in Arabidopsis as well as B.napus,it is clear that bna-miR168 a affects the combination of AGO1 and other miRNAs to form a RISC complex by regulating AGO1 negatively,then the change of “bnamiR168a-AGO1” can simultaneously mediate the expression of "bnamiR156b-SPL","bnamiR396a-GRF" and other "bnamiRNAs-Targets",further effect the flowering stage,number of branches,seed weight,SPSI and HI traits of plant.The main contents and results of this research are sa follows:1.GWAS analysis of the trait of SPSI and TSW among 588 materials in resequencing population588 resequencing population materials was planted in the base of Xiema Town,Beibei District,Chongqing City,we calculated the traits of SPSI and TSW for three consecutive years and performed the best linear unbiased estimation（BLUP）,then GWAS was performed for the mainly two traits in four environments（2017-2019,BLUP）.35 and 30 significant SNPs were detected for SPSI and TSW traits respectively,among them,the 35 SNPs of SPSI traits were distributed on 11 chromosomes in B.napus,and each of the SNP can resolve the phenotypic variation with 5.74% to 11.43%.Compared with other GWAS analysis for the traits of silique development in previous study,it is found that the SNPs located on chromosomes A01,A03,A07,and C05 are the same as the SNPs identified with the traits of seed weight and seed numbers per plant in B.napus.The significant SNPs identified for TSW traits are concentrated on the two chromosomes with C01 and A09.Each SNP locus can resolve the variation range of 6.87% to 10.08%,and 18 SNPs are the same as those identified in seed yield trait in previous study.2.Materials screening with extremely high and low SPSI as well as TSW,and transcriptome sequencing analysis among the materialsIn our study,two pairs of materials with extremely high and low SPSI [L1（46.3%）/ L2（63.3%）] and [L3（53.9%）/ L4（64.2%）] and materials with extremely high and low TSW [L5（3.9g）/ L6（6.6g）and L8（4.2g）/ L6（6.6g）] were selected from the resequencing population.The pericarps and seeds of B.napus in the early stage of nutrient accumulation（15 days after flowering）and material conversion period（35 days after flowering）were performed RNA-Sequencing（RNA-Seq）analysis.The results show that 8048,8122,7627 and 9800 differentially expressed genes（DEGs）were jointly identified in the both two pairs of SPSI materials in tissues of 15 P,15S,35 P and 35 S,respectively.5193,4702,5010 and 4456 DEGs were jointly identified in TSW materials respectively.And 10 and 7 genes belongs to DEGs identified in SPSI and TSW trait respectively were selected to verify the accuracy of the transcriptome data by q RT-PCR.The GO and KEGG analysis among the DEGs show that genes of sucrose phosphate synthase gene（BnaSPS2F）,β-amylase gene（Bna BAM4）,starch synthase gene（BnaSS2）and BnaSTP12,Bnap Glc T1,Bna ERD6-4 which belongs to MST family were all enriched in the sugar metabolism pathway,and are key genes in the synthesis and transport pathways of photosynthetic products.In order to select candidate genes that may be related to the transport efficiency of photosynthetic products accurately,we screened candidate genes in 300 kb interval upand down-stream of the significant SNPs based on the GWAS analysis of SPSI and TSW trait,further combined with the DEGs selected by RNA-Seq analysis,577 and 63 overlapping candidate genes were obtained in the two traits,respectively.And different members of the family like MST and transcription factors such as NAC and MYB were screened at the same time.12 candidate genes which expressed specificity and own similar expression pattern between two pairs of materials were finally obtained.3.Screening and identification of BnaSPS/Bna MST genes,and the results of functional analysis of BnaSPSA2-2Different members of Bn MST gene family were simultaneously identified in GWAS and transcriptome analysis method of both SPSI and TSW traits.Another key gene,BnaSPSA2-2 was identified near the S13₁783067 SNP that contributed the most in the GWAS analysis of the SPSI trait,at the same time,it was identified as DEG in the RNA-Seq analysis of L3/L4 material,and SPS was also been proved to be the key rate-limiting enzyme in synthesis of sucrose,which was the most important product of photosynthesis.Therefore,BnaSPS and Bna MST,which were both critical in the process of photosynthetic product synthesis and transport,will ready to be further functional verification in our study.At the same time,the whole genome identification of SPS and MST was carried out,and it was found that 11 and 175 members in B.napus was found in SPS and MST family,respectively,and they are separated into 3 and 7 subfamilies according to the phylogenetic tree,respectively.The overexpression and subcellular localization vector of BnaSPSA2-2 was constructed in this research,and 4 homozygous positive strains were obtained by infecting wild-type Arabidopsis,and the expression levels of BnaSPSA2-2 in the 4 strains were respectively reach 177,51,237 and 159 times compare with wild type Arabidopsis.It was found that BnaSPSA2-2 was localized on the cell membrane through subcellular localization analysis.Plants which was heterologously overexpressed of BnaSPSA2-2 showed a better phenotype than the wild type,with earlier bolting time and earlier flowering time,and the traits of number of secondary branches,number of siliques per plant,biological yield,seeds yield,SPSI and HI were all significantly higher than those in the wild type.Combined with the expression pattern of BnaSPSA2-2 among 39 tissues in ZS11 material,it shows that BnaSPSA2-2 is specifically expressed in the filaments and flower stalks at the initial and full blooming stage,as well as the stalks and siliques at the green pod stage,but almost no expression in other tissues.It shows that heterologous overexpression of BnaSPSA2-2 can accelerate the vegetative growth of Arabidopsis,leading to early flowering,and then affecting the plant’s biological yield and seed yield.4.Materials screening with extremely high and low SPSI,and miRNA sequencing analysis among the materialsBoth of the traits of SPSI and TSW are complex quantitative traits,so it is almost impossible to be fully regulated by a single gene,nevertheless,the function of that one miRNA can mediate the expression of multiple target genes at the same time,making miRNAs increasingly become the target of research on complex quantitative traits of crops.Therefore,our study performed small RNA sequencing analysis on the two pairs of materials with extremely high and low SPSI mentioned above,and finally identified 1162 new miRNAs that regulate 13177 target genes,combined with 92 miRNAs that was known in rapeseed,total of 1254 bna-miRNAs were screened in the SPSI materials.234 differentially expressed miRNAs（DEMs）was finally identified and 3405 target genes were regulated by the 234 DEMs.The q RT-PCR verification of 5 DEMs was further demonstrated the accuracy of the small RNA sequencing data in our study.Based on the results of small RNA sequencing analysis,130 and 143 DEMs were screened out of L1/L2 and L3/L4 materials respectively,and 39 common DEMs were identified in the two pairs of materials,then the KEGG analysis was performed with the 605 target genes predicted among the 39 common DEMs,it was finally determined 9 DEMs which may play a role in the transport of photosynthetic products by mediating 13 target genes involved in the sugar metabolism pathway.5.Screening and identification of Bna-miR168 a,and the results of functional analysis of Bna-miR168aIn this study,bna-miR168 a is one of the 234 DEMs we selected in the miRNA sequencing analysis,what’s more,AGO1 is both the target of bna-miR168 a and the main component of the RISC complex.Due to the particularity of miR168 and the complexity of the transport process of photosynthetic product,bna-mi168 a,which may regulate a wider range of target genes,was finally selected as the main candidate miRNA for functional verification.The overexpression and interference-expression vectors of bna-miR168 a were constructed in this study,and 4 heterologous overexpression and 4 interference-expression of bna-miR168 a homozygous positive strains were obtained after infecting Arabidopsis,and the expression level of bna-miR168 a in overexpression and interference-expression strains were 97,242,89,244 times and 0.35,0.62,0.06,0.59 times compared with the control,respectively.Meanwhile,three overexpressing and 3 interference-expression of bna-miR168 a homozygous positive strains were obtained in B.napus through the ZS11 transformation.The expression levels of bna-miR168 a in overexpression and interference-expression lines were 11,39,33 times and 0.25,0.41,and 0.51 times compared with ZS11,respectively.Heterologous overexpression of bna-miR168 a in Arabidopsis shows a negative regulatory effect that inhibits the growth and development of plants,while heterologous interference-expression plants can promote the growth and development.Plants of heterologous overexpression of bna-miR168 a show the phenotype of developmental retardation and serrated leaves,the weak vegetative growth further leads to late bolting and flowering time,what’s worse,silique abortion occurs in the reproductive growth stage significantly,resulting in a poor characters of silique number per plant,seed number per silique,silique length,seed yield,SPSI and HI.On the contrast,the bolting and flowering time of the heterologous interference-expression plants were earlier than wild-type plants,in addition,the traits of number of secondary branches,number of siliques per plant,seed yield,SPSI and HI were all significantly higher than those in wild-type plants.Meanwhile,it is confirmed that At AGO1 is the target gene of bna-miR168 a in Arabidopsis,and the expression of bna-miR403,bna-miR164 A,bna-miR156 b and bna-miR396 a between transgenic and wild-type plants was detected by q RT-PCR,the results show that the four miRNAs were all up-regulated or down-regulated by bna-miR168 a in varying degrees.Overexpression and interference-expression of bna-miR168 a in B.napus own the similar phenotypic traits to Arabidopsis.The plants in the overexpression lines performed weaker compared with ZS11,the flowering time performed later and the trait of number of secondary branches,silique number per plant,biological yield,seed yield,seed weight per silique index and harvest index are all poorer than ZS11.Interference-expression plants grow vigorously,and the trait of number of branches,silique number per plant,biological yield,seed yield,seed weight per silique index and harvest index are all higher than ZS11.It also confirmed that the three members of BnAGO1 are the mainly target genes of bna-miR168 a and bna-miR168 a negatively regulates the expression level of BnAGO1.Bna-miR403,bna-miR164 A,bna-miR156 b and bna-miR396 a were all regulated by bna-miR168 a in varying degrees.The above results show that bna-miR168 a may mediate AGO1 form a RISC complex with bna-miR403,bna-miR164 A,bna-miR156 b,bna-miR396 a and even other miRNAs,the change of "bnamiR168a-AGO1" can simultaneously mediates the expression of "bnamiR156b-SPL","bnamiR396a-GRF" and other "bnamiRNAs-Targets",and further take an impact on the traits of plant flowering,number of branches and seed yields,ultimately affect SPSI and HI of plants.