Auxin Regulates Cotton Fiber Initiation via GhPIN-Mediated Auxin Transport.

Cotton fibers are seed trichomes that make cotton unique compared with other plants. At anthesis, IAA, a major auxin in plants, accumulates in the fiber cell to promote cell initiation. However, many important aspects of this process are not clear. Here, auxin distribution patterns indicated by auxin-dependent DR5::GUS (ß-glucuronidase) expression in cotton ovules were studied during fiber cell differentiation and cell initiation [-2 to 2 DPA (days post-anthesis)]. The nucellus and fiber cell were two major sites where auxin accumulates. The accumulation in the nucellus started from -1 DPA, and that in fiber cells from 0 DPA. Immunolocalization analysis further suggests that the IAA accumulation in fiber initials began before flower opening. Furthermore, we demonstrate that accumulated IAA in fiber initials was mainly from efflux transport and not from in situ synthesis. Eleven auxin efflux carrier (GhPIN) genes were identified, and their expression during ovule and fiber development was investigated. Ovule-specific suppression of multiple GhPIN genes in transgenic cotton inhibited both fiber initiation and elongation. In 0 DPA ovules, GhPIN3a, unlike other GhPIN genes, showed additional localization of the transcript in the outer integument. Collectively, these results demonstrate the important role of GhPIN-mediated auxin transport in fiber-specific auxin accumulation for fiber initiation.

Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.

Y-shaped adaptor dependent extension (YADE) method is a useful tool to amplify the flanking sequence of a known DNA sequence, but its efficiency is frequently limited by the restriction sites around the known sequence. In this paper, we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences. With templates originating from 7 digestions, a 2,228-bp 5'-upstream sequence of a cotton small GTPase gene was obtained by two rounds of sequential YADE amplifications. The results demonstrated that the YADE method with multiple templates may be a useful tool for sequential PCR walking in complex genomes.

Transgenic cotton expressing CYP392A4 double-stranded RNA decreases the reproductive ability of Tetranychus cinnabarinus.

As a polyphagous pest, Tetranychus cinnabarinus has the ability to overcome the defense of various hosts, and causes severe losses to various economically important crops. Since the interaction between pest and host plants is a valuable clue to investigate potential ways for pest management, we intend to identify the key genes of T. cinnabarinus for its adaption on cotton, then, with RNA interference (RNAi) and transgenic technology, construct a transgenic cotton strain to interfere with this process, and evaluate the effect of this method on the management of the mites. The difference of gene expression of T. cinnabarinus was analyzed when it was transferred to a new host (from cowpea to cotton) through high-throughput sequencing, and a number of differentially expressed genes involved in detoxification, digestion and specific processes during the development were classified. From them, a P450 gene CYP392A4 with high abundance and prominent over-expression on the cotton was selected as a candidate. With transgenic technology, cotton plants expressing double-stranded RNA of CYP392A4 were constructed. Feeding experiments showed that it can decrease the expression of the target gene, result in the reduction of reproductive ability of the mites, and the population of T. cinnabarinus showed an apparent fitness cost on the transgenic cotton. These results provide a new approach to restrict the development of mite population on the host. It is also a useful attempt to control piercing sucking pests through RNAi and transgenic technology.

Functional expression of the cotton gibberellic acid oxidase homologous gene GhGA20ox1 in tobacco.

To determine the physiological function of GhGA20ox1, a homologous gene of GA 20-oxidase from elongating cotton fibers, we expressed this gene ectopically in Nicotiana benthamiana. Reverse transcription-PCR analysis showed that the GhGA20ox1 gene was expressed in the transgenic plants at various levels. It was demonstrated that overexpression of GhGA20ox1 enhanced preferentially the GA(4+7) biosynthesis in N. benthamiana and conferred GA-overproduction characters to transformants. The extent of phenotypic alteration in the transgenic plants was found to correlate with the transcriptional levels of GhGA20ox1 and GA contents. Results indicated that the GhGA20ox1 gene promoted the biosynthesis of the active GAs (GA(4+7)) in transgenic tobacco plants therefore represents a useful gene for manipulating GA levels.

The Basic/Helix-Loop-Helix Protein Family in Gossypium: Reference Genes and Their Evolution during Tetraploidization.

Basic/helix-loop-helix (bHLH) proteins comprise one of the largest transcription factor families and play important roles in diverse cellular and molecular processes. Comprehensive analyses of the composition and evolution of the bHLH family in cotton are essential to elucidate their functions and the molecular basis of cotton development. By searching bHLH homologous genes in sequenced diploid cotton genomes (Gossypium raimondii and G. arboreum), a set of cotton bHLH reference genes containing 289 paralogs were identified and named as GobHLH001-289. Based on their phylogenetic relationships, these cotton bHLH proteins were clustered into 27 subfamilies. Compared to those in Arabidopsis and cacao, cotton bHLH proteins generally increased in number, but unevenly in different subfamilies. To further uncover evolutionary changes of bHLH genes during tetraploidization of cotton, all genes of S5a and S5b 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 S5a and S5b 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 GhbHLH130D and a long terminal repeat retrotransposon inserted in GhbHLH062A, which eliminated GhbHLH062A expression in various tissues. The S5a and S5b bHLH genes of A and D genomes (except GobHLH062) showed similar transcription patterns in various tissues including roots, stems, leaves, petals, ovules, and fibers, while the A- and D-genome genes of GobHLH110 and GobHLH130 displayed clearly different transcript profiles during fiber development. In total, this study represented a genome-wide analysis of cotton bHLH family, and revealed significant changes in sequence and expression of these genes in tetraploid cottons, which paved the way for further functional analyses of bHLH genes in the cotton genus.

[The cDNA-AFLP differential display in developing anthers between cotton male sterile and fertile line of "Dong A"].

cDNA-AFLP, an effective method for mRNA differential display, was employed to compare the gene expression in developing anthers between the male sterile and fertile plants of cotton (Gossypium hirsutum L.) cultivar, Dong A. In the micro-spore stage, there were more differential bands of cDNA-AFLP than that in the meio-phase stage. Among 64 differential fragments produced by cDNA-AFLP, three were randomly selected for further analysis. RNA dot blotting showed that the GHA27 transcript was expressed mainly in floral tissues; on the other hand, the GHA28 and GHA47 transcripts were present specifically in anther. BLAST analysis demonstrated that GHA27 was highly similar to the plant ADP-ribosylation factor genes, while GHA28 and GHA27 were shown no significant similar to any sequences in the available databases.

[Cloning and characterization of a LRR resistance like (GhLRR-RL) protein gene from cotton (Gossypium hirsutum L.)].

LRR (Leucine rich repeat) proteins are involved in various biological processes. RACE was used to extend the 5' and 3' unknown sequence of a cDNA-AFLP fragment from cotton ovule, which had sequence similarity to the Arabidopsis thaliana LRR resistance-like (LRR-RL) protein. By joining the sequence of cDNA-AFLP fragment and its 3' and 5' RACE product, it was found that the full-length cDNA (GenBank Accession No. AY040532) of 1259 bp contained an ORF of 987 bp and a putative poly(A) signal. The deduced polypeptide contained 328 amino acids, and consisted of 10 LRRs with extracytoplasmic LRR consensus LXXLXXLXXLXLXXNXGXIPXX. It was demonstrated that the LRR-RL proteins of cotton and A. thaliana were highly homologous, with a distinct structure from other plant LRR proteins, suggesting that LRR-RL proteins belonged to a new LRR protein family. Using 3' RACE, the expression of LRR-RL gene was detected in root, hypocotyls, fiber, and ovules in various developmental stages.

[Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development.

[Cloning and characterization of D-113 gene promoter from cotton].

To study the expression of late embryogenesis abundant gene in seeds, the 1,024 bp 5' flanking sequence of D-113 gene, a late embryogenesis abundant gene of Gossypium hirsutum cv. Coker 312, was cloned by PCR. The similarity compared with the sequence of Lea protein gene family published was 92.50%. There are three putative ABREs and one enhancer-like which riches A/T in the promoter. The promoter was fused to the beta-glucuronidase gene to form pLD II. Via a particle bombardment, pLD II was introduced into embryogenic calli of cotton and seeds of Brassica napus which were all treated with abscisic acid for 3d before bombardment, also into roots, stems and leafs of cotton. Transient expression was measured histochemically as spot number 24 h after bombardment. GUS sexpression was observed in the seeds of Brassica napus and the embryogenic calli of cotton, but not found in roots and leaves of cotton. Those results indicated that the expression of D-113 gene promoter was embryo specific.

[Cloning and characterization of the PTS2 receptor gene (GhPex7) from cotton (Gossypium hirsutum L.)].

Peroxisomal targeting signals (PTS), including PTS1 and PTS2, were proposed to play an important role in introducing proteins into the peroxisomal matrix. Pex7, the PTS2 receptor, is crucial to the import of PTS2 proteins. Based on the sequence of a fragment (F010) recovered from cDNA-AFLP, the full-length cDNA sequence was obtained by RACE and the contig in cotton ESTs, and the coding sequence was further cloned. The GhPex7 cDNA, 1314 bp in length, contained 5 non-coding (77 bp) upstream, 3 complete downstream with polyA signal tail and ORF of 954 bp, which coded for a deduced protein of 317 amino acids. The deduced protein had a predicted MW 35.57 kDa and a pI of 5.603. Homology analysis demonstrated that GhPex7 protein contained three highly conserved domains and three G-bata domains of WD-40 proteins family. The sequence of nucleotides and amino acids shared 83% and 76% identity with known Arabidopsis AtPex7, respectively, and its amino acid sequence shared from 28% to 42% identity with those of Drosophila melanogaster, Saccach, cerevisiae, Mus musculus and Homo sapiens. Southern blotting suggested that at least two copies of GhPex7 gene existed in Gossypium hirstum genome. By Northern blotting and RT-PCR analysis, expression of the GhPex7 was detected in roots, stems, leaves, buds, ovules and fibers, however it was stronger in leaves and stems than in other tissues. At the stage of cotton's ovules and fibers development, RT-PCR analysis also indicated that expression activity of GhPex7 in ovule at 0DPA mutant plant (no-fiber) was stronger than in wild type plant, its expression in wild type fiber at 23DPA was stronger than at 12, 16 DPA too.

Isolation and characterization of a carboxylic transport protein JEN1 and its promoter from Metarhizium anisopliae.

Based on the flanking sequence of T-DNA of a T-DNA insertion mutant of Beauveria bassiana, T12, the full length cDNA of carboxylic transport protein, designated MaJen1, was cloned from Metarhizium anisopliae. MaJen1 is 1,695 bp long and contained a 1,524 bp ORF which predicted a protein of 508 amino acid. The amino acid sequence of the gene showed 69% and 31% identity to the carboxylic transport protein of Neurospore crassa and Saccharomyces cerevisiae, respectively. The genome sequence, GMaJen1, was amplified by PCR, indicating that there were two introns in GMaJen1. Southern analysis indicated that GMaJen1 was present as a singl copy in Metarhizium anisopliae. The result of RT-PCR showed that expression of MaJen1 was induced by the cuticle of cockroach and repressed by glucose. A 1,626 bp upstream sequence of GMaJen1 was amplified by YADE method, which contained several putative binding domains of glucose repressor.

[Cloning of a MADS box protein gene (GhMADS1) from cotton (Gossypium hirsutum L.)].

As a kind of transcription factors, MADS-box protein plays an important role in various cellular processes, especially in the development of floral organs. Based on the contig analysis of the cotton ESTs, the coding region of a cotton MADS-box protein (GhMADS1) was obtained by RT-PCR from floral buds of cotton (G. hirsutum). The cloned fragment of 713 bp (GhMADS1, GenBank accession no. AF538965) contains an open reading frame of 711 bp,coding a polypeptide of 236 amino acids. It was demonstrated that the deduced GhMADS1 protein was highly homologous to the AGL2 group of MADS-box proteins from Vitis vinifera, Nicotiana sylvestris, Petunia hybrida, Arabidopsis thaliana and Antirrhinum majus. Phylogenetic analysis also indicated that GhMADS1 belongs to the AGL2 group of MADS-box proteins. RT-PCR analysis showed that GhMADS1 gene expressed in petals, stamens, ovules and fibers, but not in roots, stems, leaves, bracts and sepals. The strongest expression of GhMADS1 gene was detected in petals. But in floral buds of a cotton homeotic mutant (CHV1), whose floral organs are all converted to bract leaf-like organs, the transcript of GhMADS1 gene was not detected. It was proposed that GhMADS1 gene would be crucial to the development of cotton floral organs.

[Mapping of two fertility-restoring gene for WA cytoplasmic male sterility in minghui63 using SSR markers].

F2 population derived from Shanyou63, F1 hybrid developed from the cross Zhenshan97 A/Zhenshan97B, was used in this study. Fertile bulk was constructed by polling equal amount of 15 highly fertile lines. Sterile bulk was obtained by pooling equal amount of 15 highly sterile lines. Minghui63 and Zhenshan97A, parents of Shanyou63, were analyzed with 302 pairs of SSR primers. 244 pairs of primers gave amplification products, of which 58 pairs of primers on 12 different chromosomes showed polymorphism between the two parents with polymorphic frequency up to 23.77%. Gene bulks were further assayed with the 5 pairs of primers. RM1 on chromosome 1 and RM258, RM304 on chromosome 10 was found to be polymorphic between the two gene bulks. In theory, there should be no difference detected between the two gene bulks except for the target traits governed by fertility-restoring genes. RM1, RM258 and RM304 were probably related to the restorer genes. Ten highly fertile and ten highly sterile lines were selected from F2 population of Shanyou63 to screen the gene bulks. The results showed that specific bands of Minghui63 were detected in all ten highly fertile lines while not observed in all the sterile lines. It indicated that the three SSR markers might be linked to fertility-restoring genes. Dominant lines were not selected due to their inalbility to distinguish recombinant lines from non-recombinant lines. Pure recessive lines were chosen to conduct mapping analysis. A total of 53 highly sterile lines were selected from 900 lines of Shanyou63 F2 population to estimate the genetic distance between three SSR markers and fertility-restoring genes respectively. The results demonstrated that recombination occurred in 2, 3, lines with RM1 and RM258 while no one with RM304. Using MAPMAKER/EXP 3.0, the genetic distance between RM1, RM258, RM304 and the related restorer genes were calculated as 1.9, 2.9 and 0.0 cM, respectively. It is possible that the fertility restoring gene(s) on chromosome 10 for three different types of cytoplasmic male sterility(WA, BT and HL) are of the same, or belong to a gene family.

[Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn].

Balsampear (M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method. By joining the two amplified fragments, the full-length cDNA of M. charatica homologous gene of plant class V chitinase (McChi5) was obtained. The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mochi5 gene were discussed.

[PCR walking in cotton genome using YADE method].

A Y-shaped adaptor dependent extension (YADE) method was developed to amplify the unknown genomic sequences adjacent to a known sequence, with the single-primer amplification completely suppressed. Using this method, two fragments, F027S and F027A, adjacent to a cotton ovule cDNA fragment (F027), were amplified using genomic DNA. It was demonstrated that F027S and F027A had 104 bp and 175 bp overlapped to F027, and contained 1 and 3 putative introns, respectively, all of which included conserved border sequence GT-AG and a putative branch site. The possible improvements and applications of YADE method are further discussed.

Transcriptome and biochemical analyses revealed a detailed proanthocyanidin biosynthesis pathway in brown cotton fiber.

Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs). To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols) were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form (gallocatechin and catechin), and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3', 5'-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates (dihydroflavonol 4-reductase and anthocyanidin synthase, respectively). Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3', 5'-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

Asymmetric overlap extension PCR method for site-directed mutagenesis.

Overlap extension PCR (OE-PCR) has been widely used in site-directed mutagenesis. The original OE-PCR included two rounds of PCRs and required tedious steps to purify the first-round PCR product. By combining asymmetric PCR and overlap extension, a novel asymmetric overlap extension PCR (AOE-PCR) method has been developed. This method consists of two separate asymmetric PCRs of around 30 cycles and a single cycle of annealing and extension after directly mixing the first-round PCR products. AOE-PCR eliminates intermediate purification steps and amplification of wild-type template and requires fewer PCR cycles, and is, therefore, a much simpler and faster and more efficient site-directed mutagenesis method than the original OE-PCR approach.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis.

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.