15 January 2011, Volume 23 Issue 1

    

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  The Identification and Analysis of RNA Editing Sites of 10 Chloroplast Protein-coding Genes from Virescent Mutant of Gossypium hirsutum

  JIANG 　Yuan, HE 　Jun, FAN Shu-Li, YU Jia-Ning, SONG Mei-Zhen

  Cotton Science. 2011, 23(1): 1-9.

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  RNA editing is one of the post-transcriptional regulation mechanisms of gene expression in the chloroplast of land plants. Recently, studies have shown that albino or yellow phenotype of high plants may be relevant to chloroplast RNA editing. In this paper, we investigated the RNA editing sites of 10 plasmid protein-coding genes from cotyledons and leaves of virescent mutant V1, Gossypium hirsutum, using PCR, RT-PCR and sequencing methods. There were 34 editing sites in both of them, but the editing efficiency was different in 6 sites of 34. Compared all editing sites between V1 and Coker310FR, we found that V1 had 5 novelty editing sites in accD-109, accD-468, ndhD-347, rpoA-69, and rpoA-279. Analyzed all the editing sites by using bioinformatics, the results showed that 13 sites, accD-109, clpP-187, ndhB-50, ndhB-196, ndhD-128, ndhD-225 and so on, would affect their protein secondary structures or three-dimensional structures. All the results indicate that the above-mentioned sites may play important roles in proteins assemble correctly and execute their function effectively.
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  Construction of a Fosmid Library of Chloroplast Genome in Gossypium raimondii

  LI Peng-Bo, XUE Long-Fei, WANG Yan-Xia, ZHANG 　Xi, LI Zhao-Hu, HUA Jin-Ping

  Cotton Science. 2011, 23(1): 10-14.

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  In this paper, a Fosmid library of G. raimondii chloroplast genome was constructed. The chloroplast DNA was isolated by high ionic strength and low pH buffer method. The DNA was randomly sheared and cloned into pCC1FOS vector. Recombinant DNA was packaged with the Lambda Packaging Extracts, then transfected into E. coli strain EPI300. The best sheared parameter employed in the study was 18 times with middle speed using a 1 mL injector. The library of chloroplast genome (titer: 1×10⁴ cfu·mL^-1) was obtained in which the average inserted DNA fragment was 38 kb. Thirty-nine clones covering 9.2 fold the chloroplast genome were selected by selection marker to be further analyzed. Six clones, F66, F46, F28, F8, F55, and F3, which could span G. raimondii complete genome, were screened out by cotton chloroplast markers. The library would be a valuable resource for study on genome structure and functional genes investigation in cotton.
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  Molecular Cloning and Expression Analysis of a Receptor-like Protein Kinase Gene in Upland Cotton

  YANG Yu-Wen, HE 　Bing, ZHANG Bao-Long, NI Wan-Chao

  Cotton Science. 2011, 23(1): 15-21.

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  The cDNA encoding the full length of a receptor-like protein kinase of cotton was cloned through degenerate primers amplification, genome walking and RT-PCR. Sequence analysis showed that introns were absent in the gene. It contains a 2964 bp open reading frame, encodes a deduced protein of 988 amino acid residues, and shows 60% homology to HAESA-like2, which regulates floral organ abscission in Arabidopsis. This gene was tentatively designated as GRLK5.  GRLK5 expresses higher in seed, stem bark, root, and fibre than in other organs. The ABA treatment induce the expression of GRLK5. The over expression vector was constructed by inserting GRLK5 into the pCAMBIA2301, tobacco plants were transformed by co-cultivating leaves method via Agrobacterium mediation．The object gene was verified to have been integrated into the genome of tobacco by PCR.
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  Preliminary Study on Multiplex PCR Technique and Its Application in Hybrid Cotton Seed Purity Test

  CHEN Hao-Dong, LIU Fang, WANG Wei, XIAO Cai-Sheng, LI Xiang, WANG Lin, LI Yu-Qiang, WANG Kun-Bo

  Cotton Science. 2011, 23(1): 22-27.

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  A series of stable polymorphism SSR primers in Xiangzamian series cultivars were used to construct various primer combinations according to the difference of their amplified band’s size for multiplex PCR amplification of cotton. Eighty percent of double PCR amplified normally based on the principle of amplified fragments size difference under the same conditions as their single PCR reactions. Besides, triplex and quadruple PCR were also amplified normally using primers of double PCR with good results. So we proposed a simplified protocol of multiplex PCR assay in cotton. The multiplex PCR technology was used to detect the purity of hybrid cotton seeds which clearly identified impurity and blend of female parent seeds and also other hybrid cotton seeds. It can be used as a reference for the rapid and accurate identification of cotton hybrids.
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  Effect of Regulation of Water and Nitrogen on Cotton Yield and Water Use Efficiency under Different Furrow Irrigation Patterns

  LI Pei-Ling, ZHANG Fu-Cang

  Cotton Science. 2011, 23(1): 28-33.

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  By setting the alternative furrow irrigation, conventional furrow irrigation, and fixed every-other furrow irrigation, the amount of nitrogen and irrigation design using two general rotation experiment conducted to study the regulation effect of water and nitrogen on cotton yield and water use efficiency. The results showed that: cotton yield was significantly positively correlative, with nitrogen applied amount in the range of 56.2 ~ 95.2 kg·hm^-2, and same as cotton yield and water within 37.52 ~ 160.00 mm; Cotton yield was not significant different under the same water and nitrogen treatments between alternative furrow irrigation and conventional furrow irrigation, cotton yield of conventional furrow irrigation was higher on average than the fixed every-other furrow irrigation by 9.15%. Cotton water use efficiency and nitrogen within 56.2 ~ 122.8 kg·hm^-2 was a significant positive correlation, and was significantly negatively correlated with irrigation within 37.52 ~160.00 mm. Cotton water use efficiency was no significant difference under the same water and nitrogen treatments between conventional furrow irrigation and alternative furrow irrigation, and conventional furrow irrigation was higher on average than the fixed every-other furrow irrigation in cotton water use efficiency by 9.01%. Therefore, alternative furrow irrigation can increase the cotton yield and water use efficiency.
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  Characteristics of Carbon and Nitrogen Metabolism in Two Natural Color Cotton Cultivars

  ZHANG 　Xiang, 吕Chun-Hua , LIU Xiao-Fei, CHEN 　Yuan, CHEN De-Hua

  Cotton Science. 2011, 23(1): 34-38.

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  The study was undertaken at Yangzhou University. In the experiment, the brown cotton cultivar(Xiangcaimian 2), green cotton cultivar (Wanmian 39) and white cotton cultivar(Sumian 9) were grown and investigated to study the characteristics of carbon and nitrogen metabolism. The results showed that the activity of nitrate reducatase and glutamic-pyruvic transaminase and the content of total nitrogen were lower than those of Sumian 9 before peak flowering period, but higher after peak boll period. However, indexes of the carbon metabolism(SPAD value, the content of soluble sugars, net photosynthetic rate) were always lower than those of white cotton. Further analysis results of ratio  of the content of soluble sugars to the content of total nitrogen(C/N) indicated that C/N for Wanmian 39 and Xiangcaimian 2 were lower too, that did not help the dry matter accumulation of reproductive organs.
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  Using RENI to Study the Distribution Characteristics of Cotton Roots in Different Amounts of Drip Irrigation under Film

  LIU Feng-Shan, ZHOU Zhi-Bin, CHEN Xiu-Long, GAN Yong-De

  Cotton Science. 2011, 23(1): 39-43.

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  The cotton root distribution characteristics under different amounts of drip irrigation was studied using root ecological niche index(RENI) which was compared with root parameters(root length, root dry weight, root volume, root surface area) ahead. And the results surely will be contributed to describing root distribution traits and root research methodology. The results showed: (1) RENI was well correlated with other root parameters and so can reflect root distribution characteristics. The contribution rate of root dry weight to RENI was of maximum under sufficient irrigation, and was of minimum under insufficient irrigation. However, root volume was the least influence factor of RENI under sufficient irrigation, and was the most important influence factor under insufficient irrigation. (2)Shown as skewed normal distribution, the RENI of cotton in the upper layer  soil above 40 cm depth accounted for 86.8% of the total RENI. (3) Roots developed in film-uncovered land were centrally impacted by the distance to the cotton plant，i.e., the closer of the distance，the higher of the RENI.
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  Effects of Univalent Transgenic Cotton CCRI 41 and Bivalent Transgenic Cotton CCRI 44 on Fitness and Resistance to Cry1Ac of Helicoverpa armigera

  ZHANG Yang, ZHANG Shuai, CUI Jin-jie

  Cotton Science. 2011, 23(1): 44-51.

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  The Helicoverpa armigera populations AYBC, AYBT and AYCK were fed 16 generations by transgenic cotton CCRI 41, CCRI 44 and non-transgenic cotton CCRI 49, respectively. After 26 generations of selection, compared with the control population AYCK, the larval development periods of the AYBC and AYBT populations delayed; pupal weight, fecundity and egg hatching rate reduced. Compared with the AYBT population, the larval development period of AYBC population delayed, fecundity reduced. During 17^th-26^th generations, the resistance ratios to Cry1Ac for each generation had no difference between AYBC and AYBT populations. And with generation increasing, the resistance ratios to Cry1Ac in AYBC and AYBT populations also increased.
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  Studies on Resistance Evolution of Cotton Bollworm to Bt Transgenic Cotton in Artificial Simulating "Refuges" and "Non-refuges" Conditions

  XIAO Liu-bin, SUN Hou-jun, BAI Li-xin

  Cotton Science. 2011, 23(1): 52-57.

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  n order to find out the resistance evolution of Helicoverpa armigera(Hübner) to Bt cotton, "refuges" and "non-refuges" conditions were simulated. Laboratory screening was conducted for obtaining the resistant strains of inbred and hybrid line feeding on the artificial diet which contained cotton seeds powder from Bt hybrid CCRI 29. The results showed that: screening for 11 generations, the mortality rates of inbred strain and hybrid strain of H. armigera larva decreased, larval weight and larval length increased, the larval duration shortened, pupation rate and emergence rate increased. Contrast with the hybrid strain, the inbred strain adapted faster to Bt transgenic cotton. Screening for 11 generations, using different resistant strains of cotton bollworm for transgenic insect-resistance assessment, results indicated that the resistance grade of phase-sensitive strains determination of four insect-resistant transgenic cotton cultivars did not change, and resistance grade of three transgenic varieties to self-bred lines from CCRI 29, Nankang-3, and Kemian-1 decreased by one. The resistance ratio of self-strain and hybrid strain to Bt preparations was 9.3876 and 2.5215, respectively. According to the preliminary results of this study, the policy of "refuges" to delay the resistance of cotton bollworm to Bt transgenic cotton is feasible.
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  Verticillium Wilt Resistance of  A Transgenic Cotton Line with Chitinase and Glucanase Genes

  ZHU He-Qin, FENG Zi-Li, LI Zhi-Fang, ZHAO Li-Hong, SHI Yong-Qiang, YIN Zhi-Xin

  Cotton Science. 2011, 23(1): 58-63.

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  Artificial disease nursery, severe disease field and greenhouse were jointly used for Verticillium wilt disease resistance identification of a transgenic cotton line 61217-1 into which chitinase and glucanase genes were transferred. Results showed that transgenic line 61217-1 exhibited high resistant level to Verticillium wilt disease in artificial nursery during successive three years. Average disease index of line 61217-1 was 17.9, which was significantly lower than that of the acceptor CCRI 24. In 2009, in the severe disease fields, the average disease indexes of this transgenic line were 18.7, 18.1 and 16.7 in Anyang, Henan Province, Dafeng, Jiangsu Province, and Shihezi, Xinjiang, respectively. These indexes were obviously lower than those of the acceptor CCRI 24, respectively. Furthermore, this chitinase-glucanase transgenic cotton line 61217-1 also showed higher resistant to strong virulent defoliating Verticillium dahliae, which was isolated from Hebei, Hubei and Xinjiang, respectively. And their average disease indexes were 16.4, 16.8 and 15.6, which were significantly lower than that of CCRI 24, respectively. The results indicated that the transgenic cotton line 61217-1 (Chi+Glu) exhibited excellent and stable resistance to Verticillium wilt disease.
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  Identification of the Pathogenicity-related Gene DVK1 in Verticillium dahliae

  GAO 　Feng, PENG 　Shan, PENG Xiao-Ling, LI 　Hui, LI Guo-Ying

  Cotton Science. 2011, 23(1): 64-68.

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  In this study, we carried out T-DNA insertional mutagenesis to identify mutants, which are effective in pathogenicity. The highly virulent, defoliating strain was mutated through Agrobacterium tumefaciens-mediated transformation, and 5000 transformants were obtained. Pathogenicity test of randomly selected 1000 transformants found that five mutants lost their virulence on a susceptible cotton cultivar. A new mutant, d1, was unable to cause full disease on cotton. Analysis of the mutation using Thermal Asymmetric Interlaced PCR(TAIL-PCR) confirmed an insertion into a gene DVK1. The full length of DVK1 genomic DNA and cDNA sequences were obtained using TAIL-PCR and RT-PCR methods. DVK1 has an open reading frame of 3325 bp interrupted by two introns with 52 bp and 36 bp, respectively, and putatively encodes a 1079 aa protein. The reduced pathogenic phenotype of d1 was fully complemented by reintroduction of the gene, indicating DVK1 is essential for pathogenicity in V. dahliae.
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  Dynamics Analysis of Culturable Soil Microflora and Microbial Activity in Continuous and Rotation Cropping Systems of Xinjiang Cotton

  HAN 　Jian, ZHANG Jing-Wen, XU Wen-Xiu, LUO 　Ming, WU Li-Li

  Cotton Science. 2011, 23(1): 69-74.

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  Variation of culturable soil microflora and microbial activity were investigated in continuous and rotation cropping cotton field in Xinjiang. The results showed that culturable microbial population gradually decreased with long-term continuous cropping of cotton. Compared with 5 years continous cropping, the total quantity of soil microbes in 6～8 years, 9～12 years and more than 13 years continuous cropping, decreased by 40.2%, 46.7%, 52.4%,respectively. After more than  5 years continuous cropping, the structure of the soil microbial community transformed from rich nutrition bacteria type to lower nutrition fungi type, the ratio of bacteria to fungi and actinomycetes to fungi decreased significantly. The amount of nitrogen physiological communities such as ammonifying bacteria, nitrobacteria and aerobic nitrogen-fixing bacteria decreased, while denitrifying bacteria increased. Moreover, continuous cropping resulted in soil respiration intensity and cellulolytic activity reducing. Contrary to continuous cropping, under the cotton/melilotus suavena, tomato, spring wheat or corn rotation systems were  most beneficial for increasing the total quantity of soil micro-organism, improving  the capability of soil microbial activity, adjusting the balance of microbial community. Also there was substantial increasement in the number of azotobacteria. The effects of different rotation modes were different, the benefits of cotton-tomato and cotton-melilotus suavena rotation were more obvious.
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  Study on Cold Flow Properties of Biodiesel Derived from Cottonseed Oil

  CHEN 　Xiu, LAI Yong-Bin

  Cotton Science. 2011, 23(1): 75-79.

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   The chemical compositions of biodiesel derived from cottonseed oil(CME) were analyzed by GC-MS. The cold flow properties of CME were studied by cold filer plugging point tester and crystallization mechanism of biodiesel, three approaches for improving cold flow properties of CME were put forward: (i) crystallization fractionation; (ii) blending with winter petrodiesel; and (iii) treating with cold flow improver additives. A good correlation model was proposed for predicting cold fitler plugging point(CFPP) by CME blending ratio. The study shows that the CME is mainly composed of saturated fatty acid methyl esters (C_14:0～C_24:0) and unsaturated fatty acid methyl esters (C_16:1～C_22:1, C_18:2 and C_18:3). The mass fraction of SFAME and UFAME is 32.12% and 66.19%, respectively. The CFPP of CME is 6℃. Crystallization fractionation and blending with -10PD decreased the CFPP of CME to -1℃ and -12℃, respectively. Adding not more than 1.5%(volume fraction) of Flow Fit, Flow Fit K and T818 additives decreased the CFPP of CME and CME/-10PD to 0℃ and -26℃, respectively. This study has effectively improved cold flow properties of CME and provides technical support for using CME.
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  Cloning, Sequence and Expression Analysis of Gossypium barbadense L. pepc Gene(in English)

  WANG De-Long, YU Ji-Wen, YU Shu-Xun, DI Hong-Hong, FAN Shu-Li, SONG Mei-Zhen, ZHANG Jin-Fa

  Cotton Science. 2011, 23(1): 80-89.

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  According to published EST sequences, an new pepc(Phosphoenolpyruvate carboxylase) gene was cloned from Gossypium barbadense L. cultivar 7124 by the utilization of the RACE and genome walking technology. The gene was named Gb.pepc3. It consists of 3259 bps, contains an open reading frame for 2910 bps, and encodes 969 amino acids with a deduced molecular weight of 110.7 kd and an isoelectric point of 6.08 I. Homology and phylogenetic trees analysis showed high similarity between Gb.pepc3 and pepcs in other reported plants, and all belong to C₃ type pepc. The result of the fluorescence quantitative PCR displayed that Gb.pepc3 existed in all tissues of cotton. It expressed highly in the embryo but low in fiber. The expression quantity of Gb.pepc3 varied in different stages of the development of cotton. The expression of Gb.pepc3 reached the highest level at 15 days after blossoming for Gossypium barbadense L. while 20 days after blossoming for G. hirsutum, and there was significant difference in the expression quantities between two species.
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  Effect of Acclimating at the Critical Growth Temperature on Chilling Resistance of Cotton Seedlings

  FAN Yue-Xian, WANG 　Peng, LI Sheng-Quan, LI 　Rui, LI 　Na, HAO Hui-Fang

  Cotton Science. 2011, 23(1): 90-93.

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  Chilling tolerance of cotton seedlings has been studied after acclimating at different low temperature（12℃、14℃、16℃） for 7 days and chilling stress at 4℃ for 7 days, and the possible mechanism has been discussed. The results showed that the content of proline increased in all treatments acclimating at different temperature for 7 days, among which the acclimating temperature at 12℃ was beneficial preferably, and the seedlings can tolerate chilling stress at 4℃ longer(7 days). The results approved that the chilling-resistance of cotton seedlings may be improved after acclimation at the survival threshold low temperature(12℃). Moreover, the soluble proteins in cotton seedlings were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results of the analyzing by Tanon GIS showed that the content and types of protein had changed significantly, and the new low-temperature-induced proteins(namely anti-freeze protein) were discovered at 80 kd and 90 kd in cold-hardening cotton seedlings.
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  Study on the Physiological Characters and Yield of Two Colored Cotton Varieties

  ZHANG Hai-Peng, ZHOU Qin, WANG Rong-Fu, ZHOU Tao-Hua

  Cotton Science. 2011, 23(1): 94-97.

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  In this paper, the colored cotton varieties Huaizongza 1 (brown fiber)，Huailuza 1 (green fiber）, and their parental material zms-41(the contrast) were used as experimental  materials to analyze the physiological characteristics and yield variation. The results showed the chlorophyll content (a + b) was the highest in the parental plant while lowest in green-fiber one. The chlorophyll content (a + b) were great differences between green-fiber cotton cultivar and the parental plant after full-blooming stage. Compared with the contrast, the leaf area indices of brown-fiber cultivar and green-fiber one went up mainly in the early growth stage but fell faster after full-blooming stage, and the specific leaf weight of colored cotton was relatively lower in the whole growth period. Not only the photosynthetic rate in the former full-blooming stage but also the transpiration rate in the bud stage of green-fiber cultinar and brown-fiber one were little higher than that of the contrast, but their transpiration rates were obviously lower than that of the contrast in the full-blooming stage. For yields, there were 16.7 % increase in green-fiber cultivar than in green-fiber one. Compared with the contrast, the two colored cotton varieties presented low yield, boll weight, lint percentage and number of cotton boll per plant.