15 July 2013, Volume 25 Issue 4

    

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  Tritrophic Effects of the Bt-Cry1Ah Protein on the Predator Propylea japonica

  LU Xian-Yong, ZHANG Shuai, 吕Li-Min , LUO Jun-Yu, WANG Chun-Yi, CUI Jin-Jie

  Cotton Science. 2013, 25(4): 283-290.

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  We conducted a tritrophic bioassay to evaluate the potential impact of the Cry1Ah protein on growth and the activities of development enzymes in Propylea japonica using cotton bollworm (Helicoverpa armigera) as prey. There were no significant differences in the survival rate, larval weight, larval development time, and pupation rate among P. japonica fed with cotton bollworm larvae reared an artificial diet containing Cry1Ah protein at concentrations of 0 (control), 1.0, and 10.0 μg·g^-1. The activities of some enzymes differed among the P. japonica fed with the different diets, but this did not affect the growth and development of the larvae. P. japonica fed with aphids showed significantly higher survival rate, larval weight, and pupation rate, and significantly shorter larval development time, compared with those of P. japonica fed with cotton bollworm. These results showed that different diets had a far greater effect on P. japonica than did the different concentrations of the Cry1Ah protein.
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  Cloning and Expression Analysis of an Arginine Decarboxylase Gene from Gossypium hirsutum

  LI Ya-Dong, MO Hui-Juan, WANG Sheng-Fen, SUN Yan-Xiang, MA Zhi-Ying

  Cotton Science. 2013, 25(4): 291-299.

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  Based on a sequence in a suppression subtractive hybridization library, GhADC1, GenBank accession no. KC851856, an arginine decarboxylase gene was cloned from upland cotton using bioinformatics and RT-PCR technologies. The full-length cDNA of GhADC1 was 2954-bp long. The 5' UTR was 477-bp and contained several cis-acting elements involved in stress responses and a uORF encoding an 8-amino acid sequence. The main ORF was 2181-bp long, and encoded 726 amino acids with a putative N-terminal chloroplast-targeting signal peptide. Conserved domain analysis showed that GhADC1 contained a PLP binding site and an Orn/Dap/Arg decarboxylase signal site. These characteristics indicated that it belongs to the type III PLP-dependent arginine decarboxylase protein family. Quantitative reverse transcription-polymerase chain reaction results showed that, under untreated conditions, the expression level of GhADC1 was higher in leaves than that in stems and roots, respectively, and was higher in the leaves of the resistant cotton variety Jiman 20 than that in those of the tolerant cotton variety Zhong 521. Furthermore, the expression of GhADC1 was rapidly up-regulated in the roots of the resistant variety by infection with Verticillium dahliae, while the tolerant variety did not show remarkable changes in its expression after infection. This indicated that the gene probably involved in the early defense responses to V. dahliae in the roots of the resistant cotton variety.
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  Identification of Cotton microRNAs and Their Targets by High-Throughput Sequencing

  南Wen-Zhi , WU Man, YU Ji-Wen, FAN Shu-Li, HUANG Shuang-Ling, LI Xing-Li, ZHANG Hong-Wei, ZHANG Jin-Fa, YU Shu-Xun

  Cotton Science. 2013, 25(4): 300-308.

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  miRNAs (microRNAs) are 21-24 nt non-protein-coding small RNAs that play important roles in controlling plant growth and development by directing target mRNA cleavage or translational inhibition. In this study, we constructed libraries using ovules collected at 0 DPA (days post anthesis) and 3 DPA, and fibers collected at 10 DPA, from two interspecific backcross inbred lines(BILs). The libraries were sequenced and analyzed. There were significant differences in fiber length between the two lines, while other yield and fiber quality characters were similar. By comparison with the D₅ cotton genome and other known cotton sequences, we identified 561 miRNAs, including 254 conserved miRNAs, 75 candidate conserved miRNAs, and 232 new miRNAs in the libraries. These data represent a large increase in the number of known cotton miRNAs. Most miRNAs act as negative regulators of their targets, and very few miRNAs are positive regulators. Analyses of target gene functions indicated that plant hormone metabolism was the main target for miRNAs. KEGG(Kyoto encyclopedia of genes and genomes) analysis indicates that miRNA target genes were significant enriched in plant hormone pathways.
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  Cloning and Expression Analysis of the Promoter of GhMADS29 from Cotton

  ZHANG Wen-Xiang, FAN Shu-Li, SONG Mei-Zhen, PANG Chao-You, WEI Heng-Ling, YU Shu-Xun

  Cotton Science. 2013, 25(4): 309-315.

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  We used the cDNA sequence of GhMADS29 as a search query to BLAST the genome sequences of Gossypium raimondii L. We designed the primers to clone the promoter according to the BLAST results. We obtained a 1316-bp sequence upstream from the -19 site of the start codon. Sequence analysis showed that this promoter region contained a TATA-box core element, a CAAT-box, and other cis-acting elements, including an AuxRR core, a TCA element, TC-rich repeats, an HSE, Box I, a GARE motif, and a TGA element; these elements are involved in auxin, salicylic acid, defense and stress, heat, light, and gibberellin responses, respectively. We replaced the CaMV 35S promoter in the pBI121 vector with the 1316-bp promoter sequence to construct a fusion vector containing the GhMADS29 promoter connected to the GUS gene. The fusion vector was designated as pSVP121. GUS histochemical staining analysis of transgenic Arabidopsis carrying the pSVP121 vector showed that the promoter was active in the roots and leaves of 14-day-old seedlings and in floral organs(sepals, petals, and pistils). It also showed activity in silique valves, but not in stamens and seeds. These results suggest that GhMADS29 may be regulated by light, temperature, and hormones, and it may also participate in the development of sepals, petals, and pistils. Whether or not the siliques will be dehiscent may be related to the function of GhMADS29 because of the activity of its promoter in the silique valves.
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  Effects of Controlled Release Nitrogen Fertilizer on Quality of Cotton Bolls and Fiber in Different Fruiting Branches

  LI Xue-Gang, SUN Xue-Zhen, SONG Xian-Liang, SUN Shu-Juan, CHEN 二Ying, ZHANG Mei-Ling

  Cotton Science. 2013, 25(4): 316-322.

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   The objective of this study was to evaluate the differences between urea and controlled release urea in terms of their effects on the qualities of cotton bolls and fiber. The cotton cultivar Lumianyan No. 28 was grown under field conditions with three types of controlled release nitrogen fertilizers; 100% PCU (pitch controlled urea), 50% U (urea) +50% PCU (pitch controlled urea), and 100% CRBBF (controlled release bulk blending fertilizer). Compared with the control, those treatments that received controlled release nitrogen fertilizer showed improved quality of cotton bolls and fiber. That is, they showed improved quality of cotton bolls and fiber at the third to fifth fruit node on both middle and upper fruiting branches, an increased number of fruiting branches, greater boll weight at both positions on the plant, and a decreased lint percentage. Applying controlled release nitrogen fertilizer increased the fiber micronaire, significantly improved the fiber strength, and shortened the time to reach fiber maturity, but it did not affect fiber length. These results indicated that 100% PCU and CRBBF treatments significantly affected the quality of cotton bolls and fiber.
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  Screening and Positioning of Three Chromosome-specific BAC Clones in Gossypium raimondii

  QIN 　Qin, LIU 　Fang, GAN Yi-Mei, WANG Chun-Ying, WANG Yu-Hong, CAI Xiao-Yan, WANG Kun-Bo

  Cotton Science. 2013, 25(4): 323-328.

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  We screened a bacterial artificial chromosome(BAC) library of Gossypium barbadense acc. Pima 90-53 to identify chromosome-specific BAC clones. Using BAC-fluorescence in situ hybridization technology, we obtained three BAC clones specific to chromosome D₅01, D₅02, and D₅10 of Gossypium raimondii, which could be used as cytological markers for those three chromosomes. Comparative mapping of these three BACs between G. barbadense and in G. raimondii showed that these three BAC clones could also be used to identify chromosomes D_b01, D_b02, and D_b10 in G. barbadense. The position of the BAC clone 280G06 on chromosome 10 did not show colinearity between G. barbadense and G. raimondii, possibly because of chromosomal rearrangement.
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  Effect of Light Quality on Pigment Formation in Brown Cotton Fiber

  WANG Miao, CHEN Sha-Sha, CAI Yong-Ping, LIN Yi, SUN Xu, GUO Ning, GAO Jun-Shan, JIANG Jia-Sheng

  Cotton Science. 2013, 25(4): 329-333.

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  The aim of this study was to determine the effects of light quality on pigment formation and enzyme activity in cotton fibers. The different light qualities were achieved by covering cotton bolls with different colored film filters (red, yellow, blue-violet, and white). The contents of flavonoids, total phenols, and condensed tannins, and phenylalanine ammonia lyase activity were affected by different light qualities. The different light qualities could be ranked, in terms of their effects on the content of soluble condensed tannins in fiber, as follows: red light>control>yellow light>blue-violet light. The highest content of insoluble condensed tannin in cotton bolls was those treated with red light. A correlation analysis showed that both phenols and condensed tannin contents were positively correlated with fiber pigmentation. The flavonoid content was negatively correlated with fiber pigmentation. Condensed tannins are the direct precursors of the brown pigment in cotton bolls.
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  Effect on Exogenous Coniferyl Aldehyde and Sinapyl Aldehyde on Expressions of Genes Involved in the Phenylpropanoid Pathway and Fiber Development in Cotton

  LI Xiao-Dong, YANG Yang, LI Bo, YANG Jia-Ping, FAN Ling

  Cotton Science. 2013, 25(4): 334-338.

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  We determined the effect of applying exogenous coniferaldehyde(CA) and sinapaldehyde(SA) on gene expression and cotton fiber development. The experimental materials consisted of cotton ovules in vitro. Changes in gene expression and fiber development in response to the various treatments were determined by semi-quantitative reverse transcription-polymerase chain reaction and image digitization analyses, respectively. Compared with ovules in the control, ovules treated with CA or SA at 100 and 200 μmol·L^-1 showed increased expressions of the gene encoding cinnamyl alcohol dehydrogenase(CAD), which catalyzes phenylpropanoid synthesis. They also showed increased expressions of genes upstream in the phenylpropanoid pathway, including PAL, which encodes phenylalanine ammonia lyase, C4H, which encodes cinnamate-4-hydroxylase, COMT, which encodes caffeic acid O-methyltransferase, CCoAOMT, which encodes caffeoyl-CoA-3-O-methyltransferase, and CCR, which encodes cinnamoyl-CoA reductase. High concentrations of CA and SA(200 μmol·L^-1) induced greater levels of gene expression than did the low concentrations(100 μmol·L^-1). SA induced higher gene expression levels than did CA. Both CA and SA suppressed expression of Ghexpansin1 and fiber growth; higher concentrations(200 μmol·L^-1) caused greater inhibition, and SA was more inhibitory than CA.
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  Correlation between Larval Instar and Morphological Development Indexes of Apolygus lucorum

  GAO Yong, TAN Xiu-Mei, ZHOU Hong-Xu, MEN Xing-Yuan, YU Yi

  Cotton Science. 2013, 25(4): 339-344.

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  Determination of the larval instar and its dustadiumtion is an important foundation for insect pest forecasting and management strategies. To clarify the relationship between the instar and morphological development of Apolygus lucorum (Hemiptera), we measured three morphological indexes (body length, body width, and head width) by observations under a stereo microscope. The morphological development process could be divided into three stages according to the morphological growth of A. lucorum: a rapid growth period, a slow growth period, and a stagnation period. The range of head width between neighboring instars showed the lowest degree of overlap, and its variation coefficient was lower than those of body length and body width. The growth dynamics of morphological indexes of A. lucorum were not consistent with Dyar and Crosby rules. Regression analyses of the three morphological variables against the instar showed that the exponential coefficient of determination was lower than the linear coefficient of determination. The quadratic and cubic coefficient of determination of head width against the instar showed the maximum value (R²=0.961), followed by the linear coefficient of determination (R²=0.942). The linear coefficient of body length and width against the instar was 0.940. The results of this study showed that the head capsule width of A. lucorum provides the best index to divide instars, and will be useful to accurately identify instars in research on this pest.
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  Proteomic Analysis of a Cotton Virescent Mutant Obtained by Space Mutation

  LI Hai-Jing, JIANG Bo, FAN Shu-Li, PANG Chao-You, SONG Ming-Mei, SONG Mei-Zhen, YU Shu-Xun

  Cotton Science. 2013, 25(4): 345-351.

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  We extracted total protein from the second-to-top leaves from the cotton line  CCRI 58 and its virescent mutant CCRI 58vsp, which was obtained by space mutation. The protein profiles of the two lines were obtained by isoelectric focusing followed by SDS-PAGE. We analyzed the relative abundance of differentially expressed proteins in leaves between CCRI 58 and CCRI 58vsp using ImageMaster-2D Elite 7.0 software, and further identified the differentially expressed protein spots by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/TOF) analysis. The results showed that 41 proteins showed differential expressions between CCRI 58 and CCRI 58vsp. The PI values of these proteins were mainly concentrated in the range of 4.0 to 7.0 and their molecular weights ranged from 15.0 to 95.0 kDa. After further MALDI-TOF/TOF analysis, we identified 14 of the differentially expression proteins. Among these were ribulose-1,5 bisphosphate carboxylase/oxygenase, S-adenosylmethionine synthase, and flavanone3-hydroxylase. The positively identified proteins were associated with photosynthesis and light respiration, ethylene and polyamine synthesis, and flavonoid  synthesis. Based on these differentially expressed proteins, we can explain some of the mechanisms of the cotton virescent mutant at the protein level.
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  Induction of Embryogenic Callus in Gossypium barbadense L. and Gossypium hirsutum L. by Hormone Treatments

  A Yi-Xia-Mu-Gu-Li-·Si-Ma-Yi-Li, QU Yan-Ying, SE Na-Wa-Er-·Mai-Mai-Ti-Ming, MENG Ling-Zhen, YANG Ting, WANG Li-Ping, CHEN Quan-Jia-

  Cotton Science. 2013, 25(4): 352-358.

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  The aim of this study was to induce embryogenic callus from various cultivars of cotton in tissue culture, so that a stable and efficient regeneration system could be developed to produce new cotton varieties for cultivation in Xinjiang. The explant materials were hypocotyls of the main cotton cultivars grown in Xinjiang, i. e. Xinhai 25, Xinhai 16, Xinluzao 39, and Xinluzao 42. We tested the effects of different combinations of two hormones (kinetin, KT; 2,4-dichlorophenoxyacetic acid, 2,4-D) on induction of callus from these explants. Calli were produced by the explants under four different combinations of hormones in the media. The optimal hormone combination to induce callus from Gossypium hirsutum explants was 0.1 mg·L^-1 KT + 0.05 mg·L^-1 2,4-D, while that to induce callus from Gossypium barbadense explants was 0.1 mg·L^-1 KT + 0.1 mg·L^-1 2,4-D. Hormone-free medium and medium containing double to the normal concentration of KNO₃ promoted the emergence of embryogenic callus. Filter paper placed under the medium promoted somatic embryo growth and regeneration of the root system. The differentiation and embryogenesis processes occurred more rapidly in G. hirsutum explants than those in G. barbadense explants. Using this protocol, normal plantlets of these cotton cultivars with strong roots were produced within 10 to 12 months. These methods could be used to increase the number of cotton genotypes that can be regenerated in tissue culture.
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  Effects of Smearing NAA After Decapitation on Root Activity and MDA Content of Cotton (Gossypium barbadense L.)

  BAI Deng-Sha-·Mai-Mai-Ti-Ai-Li, ZHANG Shao-Min, SUN Liang-Bin, FENG 　Gu

  Cotton Science. 2013, 25(4): 359-364.

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  A hydroponics experiment was conducted to investigate the effects of decapitation which is a conventional technique in cotton production in Xinjiang on the root activity and malonaldehyde(MDA) content of cotton under low and high nitrogen supply, respectively. The results showed that comparing with non-decapitated plants, the root activities of the decapitation, the decapitation plus smearing lanolin, the decapitation plus smearing lanolin and NAA treatments were remarkablely increased by 94.4%～110.0%, 55.9%～85.7% and 51.5%～147.4%，respectively; and the MDA content were also increased by  12.0%～17.3%, 5.2%～6.5% and 9.7%～10.9%, respectively. It is concluded that the decapitation and decapitation plus NAA smearing enhanced the growth of plant, and the activity and the total absorbing area of cotton roots.
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  Correlation Analysis of Seed Kernel Oil Content and Major Economic Traits in Cotton (Gossypium hirsutum L.)

  GUO Bao-Sheng, LIU Su-恩, WANG Kai-Hui, GENG Xiang-Li, ZHANG Xiang-Yun, GENG Jun-Yi, ZHAO Cun-Peng

  Cotton Science. 2013, 25(4): 365-371.

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  One of the new directions in cotton breeding is to develop varieties with high oil content. In this study, we analyzed the relationships between the oil content of cottonseed kernels and other selected major economic traits of cotton. We used the DPS V3.01 data processing system to calculate linear correlation coefficients using experimental data for cottonseed oil content and other agronomic characteristics obtained from 108 cotton lines. There were no significant correlations between oil content and lint yield, boll numbers per plant, boll weight, and lint percentage. There were significant positive correlations between oil content and fiber uniformity, and oil content and elongation. The positive correlation between oil content and the fiber upper half mean length was not statistically significant. There were no significant correlations between oil content and fiber strength or fiber fineness. Oil content was weakly negatively correlated with Fusarium wilt resistance, but showed no correlation with Verticillium wilt resistance. The results of this study indicated that improvement of the oil content of cottonseed kernels will not affect lint production, and only weakly affect disease resistance. The results further indicated that such improvement may improve the fiber quality to some extent. Cottonseed kernel protein content and oil content were significantly negatively correlated. The findings indicated that it will be feasible to produce new varieties with high oil contents combined with good disease resistance, high lint yield, and better fiber quality. However, it will be very difficult to breed new varieties with both high oil and high protein content.
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  Screening of Methods to Isolate High-quality Total RNA from Young Cotton Roots

  WANG Chun-Hui, ZHAO Yun-Lei, WANG Hong-Mei, CHEN 　Wei, GONG Hai-Yan, SANG Xiao-Hui

  Cotton Science. 2013, 25(4): 372-376.

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  It is difficult to obtain high-quality RNA from young cotton roots, because the root tissues contain large amounts of various secondary metabolites, including polyphenolics, polysaccharides, and tannins. Also, soil and other organic contaminants are often mixed with the roots when sampling. In this study, two commercial kits and an improved guanidine thiocyanate method were screened as methods to isolate total RNA from young cotton roots, with the aim of producing RNA of sufficient quality for RNA-sequencing analyses. The results showed that compared with the plant RNAout kits, the improved guanidine thiocyanate method resulted in greater quantities of RNA, and higher RNA quality. The RNA obtained using this method was suitable for RNA-sequencing analyses.