Screening And Functional Study Of Low Phosphorus Tolerance Genes In Brassica Napus L.

Phosphorus(P),as one of the major elements required by plants,is an important component of various organic compounds.It also participates in a series of physiological and biochemical processes and plays an important role in plant growth and development.As one of the important oil crops in China,Brassica napus provides high quality oil and also has a high economic and food value,and is extremely sensitive to phosphorus,so phosphorus has an important role in promoting the high yield and quality of Brassica napus.Plants mainly absorb phosphorus in the inorganic form in the soil,but the amount of inorganic phosphorus available in the soil is very low,while the efficiency of phosphorus utilization in oilseed rape is very low.Thus the molecular mechanism of efficient phosphorus utilization and the candidate genes of low phosphorus tolerant oilseed rape manifest advantages and provide a good understanding of phosphorus uptake by oilseed rape.In this thesis,the phosphorus-related root traits of different varieties of oilseed rape were studied by Genome-Wide Association Study(GWAS),and the detected SNP markers associated with low phosphorus tolerance and related candidate genes are all important for oilseed rape production and efficient utilization of phosphorus in oilseed rape.Meanwhile,the cloning and identification of phosphorus efficient uptake related genes,after revealing their functional properties,can further provide a theoretical basis for the selection and breeding of superior varieties to improve the yield and quality of oilseed rape in agricultural production.In this study,the important screening process was achieved by GWAS.167 different ecological oilseed rapeseeds were used as study materials,and their root morphological data(total root length,main root length,lateral root number,root surface area,root volume)were counted and used as basic data for Genome-wide Association Study to detect significantly associated SNP.The Genome-wide Association Study was used to detect significantly associated SNP markers and to set the locus range for screening candidate genes related to low phosphorus tolerance.Preliminary screening revealed that the BnaPHT1:4 protein subfamily could respond most significantly to low phosphorus.Therefore,this protein family was used as the basis for constructing an evolutionary tree as well as the determination of relative expression,and BnaC04g46050 D,which was expressed in the root system and had the highest induction of low phosphorus,was used as the target gene for functional studies.Therefore,the functional response of BnaC04g46050D(BnaPHT1:4J)made under low phosphorus conditions was identified in the second part of the study,explaining the contribution of this gene in phosphorus uptake.The preliminary identification of the target gene and the understanding of the sequence structure of the gene from bioinformatics;transgenic experiments using Agrobacterium and Escherichia coli as vector organisms to confirm the expression site and localization of the gene at the subcellular level(subcellular localization,tissue localization,RT-qPCR,etc.);phenotypic identification and ICP elemental content assay to confirm that under low phosphorus conditions,the gene In phenotypic identification and ICP elemental content measurements,we confirmed the promotion of plant growth and the contribution of the gene to phosphorus uptake under low phosphorus conditions.The main results of the study are as follows:1)It was clearly established that the root morphology of oilseed rape undergoes significant changes when phosphorus is deficient.Root morphology became a very important evaluation index in the screening process of 167 different ecotypes of Brassica napus for low phosphorus tolerance,and six varieties of Brassica napus for low phosphorus tolerance were screened.2)Screening of candidate genes.Using GWAS,17 SNP markers significantly associated with root morphological data were initially screened,including 12 associated with root length,and the corresponding candidate genes were found at 12.5 KB positions before and after the locus.By comparison of relative expression,BnaA05g06200 D and BnaA05g06230 D in the BnaPHT1:4 protein subfamily were significantly up-regulated by low phosphorus stress and had the highest expression in the root system under low phosphorus conditions.3)Identification and isolation of target gene.Since the BnaPHT1:4 protein subfamily was the most sensitive family to low phosphorus stress screened by GWAS,an evolutionary tree of this family was constructed and the expression of each gene was detected by RT-qPCR,and BnaC04g46050 D,the gene most significantly up-regulated by low phosphorus induction,was used as the target of the study.The gene sequence was cloned from a Brassica napus cDNA library,and the gene coding length was 1548 bp,coding for 516 amino acids.The gene was named as BnaPHT1:4J.4)Localization analysis of BnaPHT1:4J.The protein was detected as a transmembrane structure using the protein structural domain prediction tool.To further understand the localization of BnaPHT1:4J,expression vectors of p35s::BnaPHT1:4JGFP and p BnaPHT1:4J::GUS were constructed during the study for subcellular localization and tissue localization,respectively,and observed under microscope.The subcellular localization results showed that the protein was localized to the plasma membrane,and the staining results of GUS indicated that BnaPHT1:4J was specifically expressed in roots and the signal intensity was induced by low phosphorus.The RT-qPCR assay of BnaPHT1:4J was carried out on root,stem and leaf tissues of rape under different phosphorus treatment conditions,which further confirmed that BnaPHT1:4J was mainly expressed in the root system and its expression was significantly up-regulated by low phosphorus induction.5)Validation of phosphorus transport function of BnaPHT1:4J.The BnaPHT1:4J overexpression vector was introduced into phosphorus transport-deficient mutant brewer’s yeast(Δpho84)by yeast transformation,and the results showed that colonies of the BnaPHT1:4J overexpression strain were significantly larger than those of the wildtype and mutant strains under 0.1 mM Pi.6)Heterologous overexpression of BnaPHT1:4J in Arabidopsis thaliana in response to low phosphorus stress.The BnaPHT1:4J overexpression vector was introduced into Arabidopsis thaliana by transgenic technique and transgenic Arabidopsis thaliana was obtained after positive identification.Wild-type,overexpression lines,mutant atpht1:4 and complementary lines were used as materials for phenotypic identification experiments.The four types of lines were subjected to both low and high phosphorus treatments and root morphology was observed.The results showed that the main root length of the BnaPHT1:4J overexpression lines and the complementary line were significantly longer than that of the wild-type and the mutant,respectively,under low phosphorus conditions.7)Phenotypic analysis of BnaPHT1:4J overexpression plants under low phosphorus stress conditions.The BnaPHT1:4J overexpression vector was introduced into Brassica napus by transgenic technique and the transgenic oilseed rape was grown by tissue culture technique and obtained by positive identification.Similar to the phenotypic identification of Arabidopsis thaliana,the values of main root length,leaf area,root system,and above-ground fresh weight,and the phosphorus content in the root system were significantly higher in the transgenic plants under low phosphorus conditions than for the transformed lines.In addition,the contents of potassium(K),magnesium(Mg),iron(Fe),zinc(Zn),manganese(Mn)and sodium(Na)were also varied under low phosphorus.8)BnaPHT1:4J affects the expression of genes related to phosphorus uptake.By RT-qPCR,it was found that BnaPHT1:4J homologs,BnaPHR2 and some key enzyme genes in gibberellin biosynthesis were differentially up-regulated in BnaPHT1:4J overexpression lines.In summary,this study detected a series of SNP loci associated with root phenotypes subjected to low phosphorus stress based on Genome-wide Association Study of phosphorus,and several candidate genes associated with low phosphorus tolerance were screened by gene information within SNP differential loci,and BnaPHT1:4 was identified as a gene family subjected to low phosphorus stress.In-depth studies revealed that BnaPHT1:4J in BnaPHT1:4 protein subfamily is a phosphorus uptake related transport protein with high affinity for phosphorus that is specifically highly expressed in the root system of Brassica napus and is most significantly induced up-regulated by low phosphorus stress,which plays an important role in phosphorus deficiency stress and may also have positive regulation of gibberellin biosynthesis to promote the growth and development of oilseed rape.This study provides important theoretical support for future screening of phosphorus-efficient oilseed rape germplasm.