| Anthocyanin is an important plant pigment which is accumulated in flowers,fruits and leaves.The anthocyanins in flowers and fruits play a role in attracting pollinators and seed dispersers in plants.The anthocyanins accumulated in the leaf are induced by sunlight and other stresses.Its biological function is to protect plants from ultraviolet rays,low temperature,pathogen invasion and herbivore feeding.Red plant(R1)gene is a traditional genetic marker in cotton.R1 cottons exhibit red to purple color in leaves,stems,petals and bolls due to the accumulation of anthocyanins.It has the advantage of insect resistance.Sub-red plant(Rs)cottons showed similar empire red leaves,but the pigmentation in Rs tissues is much lighter compared to that in R1.Interestingly,Rs leaves showed significantly higher photosynthesis efficiency compared to R1 and normal cottons.Therefore,genetic research on upland cotton R1 and Rs materials have an important theoretical and practical value for improving cotton quality.The upland cotton variety T586 contains red plant(R1)gene,and the genetic background is quite different from Yumian 1 material.The previous study construct a high-density genetic map of upland cotton used SSR molecular markers and T586 × Yumian 1 recombinant inbred lines(RIL)population.However,the number of SSR markers between the two materials is close to saturation,and the use of these SSR markers cannot meet the requirements for fine mapping or map-based cloning of specific trait genes in the two materials.With the publication of the genome of upland cotton,genome resequencing technology has made it easier to develop SNP markers and In Del markers for upland cotton,which facilitates map-based cloning of quality traits.In this thesis,by re-sequencing the genomes of the upland cotton cultivars T586 and Yumian No.1,a large amount of SNP and In Del loci information between the two upland cotton materials were obtained.The positional relatedness of Gh PAP1 D genes to R1 phenotypes was further evaluated genetically using a recombinant inbred line(RIL)population of T586 × Yumian 1 with 8 newly developed markers.Along with transcription analysis and genetic transformation,the anthocyanin regulation mechanism of Gh PAP1 D in cotton was explored.In addition,the gene function of the anthocyanin regulatory gene of the sub-red(Rs)plant mutant was also studied.With evidence from RNA-Seq,comparative cloning and genetic linkage analysis,and knockdown by virus induced gene silencing(VIGS),we demonstrated that the Rs phenotype resulted from moderate upregulation of Gh PAP1 A,which is the homolog of the R1 gene.The 50 bp tandem repeats in the promoter region of Gh PAP1 A was the reason for the up-regulation of Gh PAP1 A in Rs plant.The main results are as follows,1.The SNP and In Del difference information between the upland cotton material T586 and Yumian 1 was obtained by whole genome resequencingWhole genome resequencing of upland cotton cultivars T586 and Yumian 1 were performed.In total Clean Data of 31.42 Gbp and 29.55 Gbp were obtained respectively,with an average sequencing depth of 10×.Quality control of the sequencing data found that the quality of the sequenced bases was high(Q20>= 96.21%),and the GC content was between 36.53% ? 37.07%.So the amount of data obtained through sequencing and the sequencing quality of the two samples were qualified.By aligned to the upland cotton reference genome assembly of cv.TM-1,it is found that the aligned rate of the two samples reads is between 98.89% and 99.53%,and the average coverage depth is between 10.65 X and 13.19 X,indicating that the alignment results are normal and can be used for follow-up SNP and In Del mutation detection.SNPs detection on the two samples showed that a total of 2,288,133 SNP loci were detected in the T586 sample,and 1,890,741 SNP loci were detected in Yumian 1.The most of the SNP loci were distributed in the intergenic region(2,014,437 and 1,678,640),followed by the intron region(117,309 and 88,167 respectively).In addition,a statistical comparison of the SNP data between the two samples found that there are 2,131,593 SNP differences between the two materials.Analysis of the SNP mutation frequency found that the transions is greater than the transversions mutation(ts/tv=2.2).The large number of SNPs obtained is beneficial to the development of corresponding molecular markers.In Del loci were also statistically analyzed in the two samples.Relative to the reference genome,191,876 and 130,718 In Del loci were obtained respectively,which were mainly distributed in the intergenic region(151,087 and 104,163),followed by the intron region(15,357 and 10,101).Statistics of In Del sites found that there were 184,801 In Del sites between the two samples.Counting the length of In Del in the coding region,it is found that the number of In Del gradually decreases with the increase of its length,and the number of In Del with a multiple of 3 length is higher than its both sides.It is speculated that it will not cause frameshift mutation therefore can be retained in evolution.2.Genetic mapping,sequence analysis and functional verification of the R1 geneUse the SNP and In Del data obtained by resequencing to develop SNP markers in the R1 gene region.Among them 8 SNP markers have good polymorphisms among the parent materials.Use the RIL populations of T586 and Yumian 1 to locate the R1 gene to the newly developed SNP markers S5 and S6,and the physical distance in the upland cotton genome is about 232 Kb contains 3 annotation genes.Transcriptome analysis found that only the expression level of Gh PAP1D(Gohir.D07G082100)was different among the three genes.Sequence analysis found that the Gh PAP1 D promoter region in the two materials contained a 228 bp tandem repeat difference,which was consistent with the previously reported RLC gene,thus proving that Gh PAP1 D is the control gene of the red plant R1.The genetic transformation of Gh PAP1 D in tobacco and cotton proved that the anthocyanin content of the Gh PAP1 D gene overexpressing cotton and tobacco materials increased significantly.The leaf color changed to red,which proved that Gh PAP1 D can enhance the gene function of anthocyanin synthesis and accumulation;In addition through the VIGS method down-regulating the expression of Gh PAP1 D in R1 red plants cotton,it was found that the color of cotton leaves changed to green,and the anthocyanin content also decreased accordingly.The results confirming that Gh PAP1 D in cotton leaves played a function of regulating anthocyanin synthesis.In order to clarify how Gh PAP1 D regulates anthocyanin synthesis in cotton leaves,transcriptome sequencing was performed on the Gh PAP1 D overexpressing transgenic cotton and its Null line.A total of 567 differentially expressed genes were found between the two materials,of which 305 were up-regulated genes and 262 were down-regulated genes.Among the differential genes,38 genes related to anthocyanin synthesis were upregulated,and no down-regulated genes related to anthocyanin synthesis were found.Five structural genes related to anthocyanin synthesis were also significantly up-regulated in R1 plants.The dual luciferase reporter system was used to detect the interaction between Gh PAP1 D and the promoters of downstream structural genes,and it was found that Gh PAP1 D could transcriptionally activate the promoters of Gh UFGT and Gh GST.In addition,the insect resistance of the red leaves overexpressing Gh PAP1 D was tested.The results showed that cotton leaves overexpressing Gh PAP1 D not only affect the feeding of the cotton bollworm,but also inhibit its growth and development,so it has insect resistance to the cotton bollworm.The insect resistance test of spider mites also found that it inhibited the development and reproduction of spider mites.It shows that Gh PAP1 D can be modified by genetic engineering methods to obtain cotton materials with broad-spectrum insect resistance of two different mouthpart types.3.Expression level analysis,sequence analysis and functional identify of anthocyanin regulatory gene Gh PAP1 A in sub-red(Rs)plantThe sub-red(Rs)plant and the red plant R1 have similar phenotypes.Both the leaf color changes to red,but its color is lighter than that of the red plant R1.It is found that the anthocyanin content has increased significantly by measuring the anthocyanin content.Therefore,transcriptome analysis was also performed on the sub-red plant,in which the expression changes of genes related to the anthocyanin synthesis pathway were analyzed.Similar to the results in the red plants,the expression levels of the five structural genes related to anthocyanin synthesis also increased relative to the green plants,but the R1 did not increase as much as the red plants.In addition,the detection of the regulatory genes of the anthocyanin synthesis pathway found that the expression of Gh PAP1 A increased significantly,while the expression of Gh PAP1 D did not change,so Gh PAP1 A may be the controlling gene of its sub-red phenotype.The sequence analysis of the Gh PAP1 A gene revealed that lot of differences of SNP and In Del in the promoter region of the sub-red plant and the green plant.In addition,comparing the responding Ga PAP1 sequence of Gossypium arboreum,it was found that the coding sequence of the sub-red plant was exactly the same as that of Gossypium arboreum.Only a 50 bp tandem repeat difference was found in the promoter region,indicating that the Gh PAP1 A gene in the Rs plant was recently introgressed from Gossypium arboreum.GUS staining found that the promoter activity in Rs plant was higher than that in Gossypium arboreum,indicating that the 50 bp tandem repeat difference in the promoter region caused the difference in gene expression.The coding region of the Gh PAP1 A gene in the Rs plants and green plants has 3 missense mutations.To compare the biological function of Gh PAP1 A from Rs and GL cotton,these two genes were overexpressed in tobaccos.It was observed that the phenotypes of the two tobacco transformants were the consistent,both of the leaf color changed to red,and the anthocyanin content was significantly increased compared to wild-type tobacco,but there was no difference between the two transformants.Therefore,it is proved that although the coding region of the Gh PAP1 A gene in the sub-red plant and the green plant is different,they all have the same function of promoting anthocyanin synthesis.It is further confirmed that the phenotypic difference between the sub-red plant and the green plant is caused by the difference in the promoter region of Gh PAP1 A.In addition,the VIGS method was used to down-regulate the expression of Gh PAP1 A in the sub-red plant material,and it was found that the red leaves of the sub-red plant material changed to green,and the anthocyanin content was also significantly decreased,thus further confirming that Gh PAP1 A regulates anthocyanin synthesis in the sub-red plant material. |
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