Map-based Cloning Of Dark Brown Fiber Gene Lc₁,And The Regulation Of Proanthocyanidin Biosynthesis And Brown Fiber Coloration

Naturally colored cotton synthesizes and accumulates pigments during the development of cotton fiber, and is the major source of naturally colored fibers. With natural colors, naturally colored cotton by-passes dying, decreases the costs of energy and water in textile processing, and avoids the residual toxic organic dyes and inorganic ions in textiles. Recent years, the naturally colored cotton and its fabric are increasingly attractive to the consumption with increasing awareness of environmental protection and green consumption. However, the development of naturally colored cotton industry is obstructed due to low yield, short fiber length and limited colors of available germplasms. Brown fiber cotton is the most widely-used naturally colored cotton at present. Researches have shown that the brown fiber gene is controlled by Lc1, and the pigment in brown fiber belongs to proanthocyanidins(PAs), a class of flavonoids. However, there was no report that Lc1 was cloned successfully, and the regulatory mechanisms of PA biosynthesis is still unclear in brown fiber. Therefore, revealing the regulatory mechanism of pigment synthesis and color generation in cotton fiber provides a theoretical basis to improve the yield and quality of naturally colored cotton, which is also of great significance to promote the development of China’s cotton industry. In higher plants, the synthesis of PA is co-regulated by MYB-b HLH-WD40 ternary complex. In the brown seed coat of Arabidopsis thaliana, the three types of transcription factors involved in the regulation of PA synthesis are TT2, TT8 and TTG1, respectively. Among them, TT2 plays a crucial role in the synthesis of PA in A. thaliana seed coat, which can independently activate the expression of downstream structural gene. TT8, a subfamily III b HLH transcription factor of A. thaliana, can combine TT2 and significantly enhance the activation effect on the downstream target gene. To study the regulatory mechanism of PA synthesis and fiber coloration in cotton, TT2 and TT8 homologous genes in cotton were identified in this paper. The relationship between TT2 and TT8 homologous genes and the synthesis of PA and the coloration of brown fiber was revealed by expression analysis, transgenic cotton, transient expression in tobacco, and fine mapping of brown fiber gene Lc1. The main results are as follows,1. Cloning, functional verification, and expression analysis of cotton TT2 homologous genesWith the TT2 amino acid sequence as a probe, we yielded 21 similar proteins in the search of Gosspypium.raimondii genome. Further phylogenetic analysis shows that 5 proteins(Gorai.001G015200, Gorai.010G087200, Gorai.001G020600, Gorai.001G020400, and Gorai.001G020500) are as TT2 homologs in G. raimondii clustered with TT2 of A. thaliana and named as Gr TT2-1~ 5, respectively. Five pairs of Gh TT2 were cloned from the A and D subgenome of the brown-fiber upland cotton T586. Sequence analysis showed that the structures and sequences of orthologous Gh TT2 genes originated from different subgenomes were highly conserved, while the paralogous genes diverged more in sequences.To verify the function of these Gh TT2 genes, 4 Gh TT2s(Gh TT2-1D~5D, except for Gh TT2-3D) in D subgenome and Gh TT2-3A were constructed downstream to a constitutive promoter(35S), and transformed into white-fiber cotton. The result indicated that transgenic cotton calli could be stained blue by DMACA(PA specific stain). In addition, the PA contents in these calli were 5-10 fold higher than the wild type. It suggested that these Gh TT2 s all had the function to promote PA biosynthesis.The expression level of 10 Gh TT2 s in various tissues and fiber at different development stages of white-fiber and brown-fiber upland cotton were detected by q RT-PCR. Results showed that two pairs of Gh TT2s(Gh TT2-1A/D and Gh TT2-2A/D) had the similar expression pattern, were preferentially expressed in cotton ovules. Two pairs of Gh TT2s(Gh TT2-3A/D and Gh TT2-4A/D) showed different expression patterns in the cotton tissues. Gh TT2-3A was highly expressed in the fiber of brown cotton. Gh TT2-3D preferentially expressed in white-fiber cotton ovule. Gh TT2-4A mainly expressed in the petal of brown-fiber cotton. Gh TT2-4D mainly expressed in the leaf of white-fiber cotton. Gh TT2-5A/D had no expression in any tissues. Except Gh TT2-3A, the other 9 Gh TT2 genes does not expressed in the developing fibers. Gh TT2-3A expressed highly in the different developmental stages of brown fiber, and PAs were consistently synthesized and accumulated in these fibers. These results showed that the 10 TT2 homologous genes have different expression pattern in cotton tissues.The expression level of these 10 genes were further detected in developing fibers of brown-fiber and white-fiberrecombinant inbred line(RIL). The results suggested that Gh TT2-3A had high expression in brown fibers but not in white- fibers. The expression of the rest 9 genes were hardly detected in the two kinds of cotton fibers. It was indicated that Gh TT2-3A was specially expressed in brown fiber, which may take part in the regulation of brown pigment synthesis.2. Gh TT2-3A is a candidate gene for brown fiber(Lc1)The previously research have shown that lint color 1(Lc1) up-regulated the whole PA pathway and promote the coloration of brown fiber. To clarify the relatedness of Gh TT2-3A to Lc1 and brown fiber coloration. I used 2 populations to map Lc1. A Gh TT2-3A-BAC was screened from T586 BAC library, to search the molecular marker linked with Lc1. Based on sequences of Gh TT2-3A-BAC and the surrounding genomic sequences, a series of SSR primers were designed. SSR markers Lc08 and Lc-FT3 linked with Gh TT2-3A were screened. Further, by comparing cloning sequences from parents T586 and Yumian No. 1, six SNP or indel markers(PHDA, MSF10A-3Y, TT2-3A3 Y, TT2-4A, ERE, and Pec53l) were screened. A mapping population of 270 RILs derived from T586 × Yumian 1 and 1698 individuals of F2 plants were genotyped with 8 markers for fine mapping. In the RIL population, Lc1 was mapped between MSF10A-3Y and Pec53 l, with a genetic distance of 0.3 c M. In the F2 population, Lc1 located between the markers TT2-3A3 Y and TT2-4A which had a genetic distance of 0.7 c M. The target interval was 47 kb between TT2-3A3 Y and TT2-4A, containing two coding genes Gh TT2-3A and Gh TT2-5A. Combined with the previous expression results, I inferred that Gh TT2-3A was the candidate gene for Lc1, and may take part in the regulation of brown pigment synthesis.3. Overexpressing the Gh TT2-3A at the secondary cell wall biosynthesis stage is key to coloration of brown fiber.To clarify the regulatory role of Gh TT2-3A on the PA synthesis in cotton fiber, we carried out a functional analysis by transgenic method. Gh TT2-3A was constructed downstream to constitutive promoter 35 S and fiber–specific promoters of different developmental stages(SCFP, E6, and Fb L2A). Genetic transformation of white-fiber cotton was performed via Agrobacterium mediated method. Results showed that the expression level of Gh TT2-3A was obvious up-regulated in the early developmental stage(11DPA) of transgenic cotton of SCFP::Gh TT2-3A, E6::Gh TT2-3A, and 35S::Gh TT2-3A. At the same time, the content of PA in fiber was 1-2 fold higher than that of wild type. While the expression level of Gh TT2-3A was significantly decreased in late developmental stage of fiber and the content of PA in the fiber was almost no different from that in the wild type. The situation in Fb L2A::Gh TT2-3A transgenic cotton was just opposite and it’s mature fiber was obviously brown. Gh TT2-3A in early development(11DPA) of Fb L2A::Gh TT2-3A transgenic cotton fiber had almost no expression and the PA content in the fiber also had no change than the wild type. However, with the advent of the secondary wall synthesis period, the expression of Gh TT2-3A in the fiber was gradually up-regulated andthe amount of PA synthesis and accumulation in transgenic fibers was significantly increased. These results indicated that Gh TT2-3A had the function of promote the synthesis of PA in the transgenic cotton fibers, and the synthesis and accumulation of PA in the late stage of fiber development(secondary wall synthesis) plays the key role in the process of presenting color in mature fiber.To verify the downstream targets the influence of Gh TT2-3A, Digital Gene Expression Tag Profiling analysis of 22 DPA fibers of transgenic cotton(Fb L2A:: Gh TT2-3A) and Null(control) was performed. It turned out that compared with Null, a total of 170 differentially expressed genes were identified in the transgenic cotton, among which 149 were up-regulated and 21 were down-regulated. A total of 37 PA synthesis pathway genes were identified, and the expression of these genes was significantly up-regulated. They include 5 Gh PALs, 4 Gh C4 Hs, 2 Gh4 CLs, 5 Gh CHSs,4 Gh CHIs, 3 Gh F3 Hs, 4 Gh F3’Hs, 4 Gh F3’5’Hs, 2 Gh DFRs, 3 Gh LARs, 2 Gh ANSs, and 2 Gh ANRs. Further RTPCR analysis suggested that expression of these genes in Fb L2A::Gh TT2-3A transgenic cotton fiber were much higher than Null line. In addition, 2 Gh WD40 s and 1 Gh TT8 homologous genes were up-regulated more than 3 folds. These results showed that Gh TT2-3A up-regulated the whole PA synthesis pathway in transgenic fiber, and also can activate the transcription factors involved in regulation of PA.These results indicated that Gh TT2-3A promoted the synthesis and accumulation of PA in transgenic fiber by activating the whole PA pathway, and finally the mature fibers appeared brown color. Meanwhile according to the prevous expression and genetic location analysis, I can make sure that Gh TT2-3A is the brown fiber gene Lc1 which determining the synthesis of brown pigment PA.4. Identification of cotton TT8 homologous genesBased on the previous study, we further determined the TT8 humongous gene related to PA biosynthesis in cotton. By searching b HLH homologous genes in sequenced diploid cotton genomes(G. raimondii and G. arboreum), a set of cotton b HLH reference genes containing 289 paralogs were identified and named as Gob HLH001-289. Based on their phylogenetic relationships, these cotton b HLH proteins were clustered into 27 subfamilies. Compared to those in Arabidopsis and cacao, cotton b HLH proteins generally increased in number, but unevenly in different subfamilies. Among those b HLH proteins, the S5 a and S5 b b HLH subfamilie showed highest sequence similarity to conserve with the TT8. In S5 a and S5 b subfamilie. The number of cotton b HLH included Gob HLH062, Gob HLH064, Gob HLH110, Gob HLH123, and Gob HLH130, respectively.To further define the gene structure of S5 a and S5 b b HLH genes in upland cotton. All genes of S5 a and S5 b subfamilies in upland cotton and its diploid progenitors were cloned and compared, and their transcript profiles were determined in upland cotton. A total of 10 genes of S5 a and S5 b subfamilies(doubled from A- and D-genome progenitors) maintained in tetraploid cottons. The major sequence changes in upland cotton included a 15-bp in-frame deletion in Ghb HLH130 D and a long terminal repeat retrotransposon inserted in Ghb HLH062 A.5. Gh TT2-3A and Ghb HLH110 A synergistically activate the PA pathwayTo elucidate the regulatory mechanism of Gh TT2-3A and b HLH involved in PA biosynthesis, the Gh TT2-3A and Ghb HLH110A’s activation effects on Gh DFR, Gh LAR, and Gh ANR promoters were test by transient promoter activation assays in Nicotiana benthamiana. The results showed that both Gh TT2-3A and Ghb HLH110 A alone were able to weakly activate the Gh DFR, Gh LAR, and Gh ANR promoter, when the two cotransfected, the activation effects were significantly enhanced. These results suggested that Gh TT2-3A and Ghb HLH110 A had a synergistic effect on regulating PA structural genes.In this study, I identified 5 pairs of Gh TT2 homologous genes which can promote PA in cotton. Gh TT2-3A specifically expressed in brown fibers, and promoted PA synthesis and accumulation at different stages of fiber development. But the brown coloration in mature fibers presented only when Gh TT2-3A was expressed in the fibers of secondary cell wall synthesis stage. Based on the result of genetic mapping, I confirmed that Gh TT2-3A is the lint color gene Lc1, and obtained the transgenic brown-fiber cotton. Gh TT2-3A and Ghb HLH110 A could synergistically activate PA biosynthesis pathway. This study laid the foundation for studying the molecular mechanism of pigments’ effects on fiber development, and provided a new material for colored cotton breeding.