15 September 2013, Volume 25 Issue 5

    

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  Analysis of Transgenic Arabidopsis thaliana with the Gossypium hirsutum L. MADS- box Gene GhMADS13 

  JIANG Su-Cheng, SONG Mei-Zhen, PANG Chao-You, WEI Heng-Ling, FAN Shu-Li, YU Shu-Xun

  Cotton Science. 2013, 25(5): 377-381.

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  GhMADS13, whose primer designed according to submitted sequence, was obtained by PCR with the 100% identity to explore its function. qRT-PCR in different tissues revealed that GhMADS13 had the highest expression level in the flower, and this was hundreds of times greater than root with the lowest expression level. GhMADS13 was expressed in all floral organs, and expression levels in the sepal, petal, stamen, carpel, ovule were little different, and ovule had the highest expression level. GhMADS13 was inserted into a pBI121 vector, and a plant over-expression vector was constructed successfully. Two lines of transgenic Arabidopsis were obtained by floral-dipping. The results of the molecular detection and phenotypic data showed that the Arabidopsis plants with high expression levels of GhMADS13 were shorter, and produced less seeds and siliques. Based on the qRT-PCR results and the analysis of ectopic expression, we deduce GhMADS13 mainly negatively regulates ovule development.
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  A New Method for Identification of the Genetic Purity of Cotton Varieties by SSR Markers

  LIU Guo-Dong, WANG Fu-Rong, GONG Yong-Chao, MA He-Huan, ZHANG 　Jun

  Cotton Science. 2013, 25(5): 382-387.

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  To establish a method for genetic purity identification of cotton (Gossypium hirsutum L.) varieties by SSR markers, marker genotypes of 12 conventional cotton genotype varieties were surveyed using 78 pairs of core SSR primers. By comparing SSR loci from different individuals of all tested cotton varieties, the non-homozygous SSR alleles of cotton variety were divided into three typical scenarios at the molecular level. Consequently, a method for the genetic purity identification of cotton varieties using SSR markers was proposed based upon analysis of the comprehensive influences of both non-homozygous SSR loci and the rate of heterotype individuals in the cotton varieties. For the genetic purity of 12 cotton varieties tested by this method, three varieties (C5, C9 and C11) were above 98%, while two varieties (C3 and C6) were 67.31% and 31.79%, respectively. The method proposed takes into account both the influences of farraginous individuals detected by SSR markers and the inherited non-homozygous SSR loci on the purity of a cotton varieties, and therefore, provides an effective and reliable way to identify the uniformity and stability of cotton varieties.
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  Response of Three Resistance Genes to Damage of Aphis gossypii Glover (Hemiptera: Aphididae) in Cotton

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

  Cotton Science. 2013, 25(5): 388-395.

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  Cotton has a complex genetic background. The aim of this study is to better understand the responses of resistance genes in different cotton varieties damaged by Aphis gossypii. We collected damaged leaves in the field from four cotton varieties, CCRI 41, CCRI 44, CCRI 49 and CCRI 79. Fuorescent quantitative PCR, was used to analyze the expression levels of polyphenol oxidase(PPO) genes, Cu/Zn superoxide dismutase (Cu/Zn SOD)  and heat shock protein70(HSP70) genes. The results showed that the responses of these resistance genes to damage of A. gossypii were different in the four cotton varieties. Thiis indicates that different cotton varieties use different resistance networks to response aphid damage. Our findings provide a reference for selecting aphid resistant cotton, and for the study of resistance mechanisms.
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  Expression, Purification and Enzymatic Activity of a Membrane-bound Trehalase Gene from Apolygus lucorum

  TAN Yong-An, XIAO Liu-Bin, BAI Li-Xin, SUN 　Yang

  Cotton Science. 2013, 25(5): 396-402.

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  Using a previously obtained cDNA sequence encoding a membrane-bound trehalase from Apolygus lucorum, a pET28a vector containing ALTre-2 gene was constructed and transformed into BL21 competent of E.coli. The target recombinant protein was overexpressed, and purified using Ni-NTA (Nickel-nitrilotriacetic acid)  agarose, and then its activity and enzymatic characteristics were studied. The results showed that the ALTre-2 gene could be overexpressed in E.coli, and the target recombinant protein had a higher trehalase activity (84.747±3.299 nmol·μg^-1·min^-1) when using trehalose as substrates; the most suitable reaction temperature was 50 ℃, and the ideal pH was 7.0. Our results facilitate further molecular research on the membrane-bound trehalase from Apolygus lucorum.
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  Effects of Nitrogen Concentrations on Cotton(Gossypium hirsutum L.) Seedling Growth and Photosynthetic Characteristics

  CHEN Jing, LIU Lian-Tao, SUN Hong-Chun, ZHANG Yong-Jiang, WANG Zhan-Biao, LI Cun-Dong

  Cotton Science. 2013, 25(5): 403-409.

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  Cotton seedling growth and photosynthesis and chlorophyll fluorescence indexes under different nitrogen concentrations were determined in indoor solution cultures. The results revealed that dry matter accumulation, chlorophyll (a+b), chlorophyll a/b, net photosynthetic rate, stomatal conductance, transpiration rate and fluorescence characteristics of cotton seedlings at first increased and then decreased with the increase of nitrogen concentration. The most suitable nitrogen concentration for cotton seedling growth was 4 mmol·L^-1. The highest significant correlation between the indexes and nitrogen concentration was observed after 12 days of treatment. The most sensitive indexes were those of stomatal conductance and net photosynthetic rate. The photosynthesis indexes in Lumianyan 28 decreased more than those of Yinrui 361 and Nongdamian 8. Lumianyan 28 was the most sensitive variety, Nongdamian 8 the least sensitive variety, Yinrui 361 was mid-range.
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  Studies on the Genetic Transformation Method by Cotton Shoot Tip-Agrobacterium Medium Using the Glyphosate Resistant Gene

  OU Ting, HE Qiu-Ling, CHEN Jin-Hong, ZHU Shui-Jin

  Cotton Science. 2013, 25(5): 410-416.

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  A genetic transformation system via cotton shoot tip-agrobacterium medium based on a glyphosate resistant gene was established through the optimization of the glyphosate concentration during screening, infection period and infection concentration of the agrobacterium, period of co-culture with the agrobacterium, and the conditions of recover culture etc., used CCRI 49, Coker 201, and YZ-1 as the materials, and their transgenic herbicide resistant cotton plants with EPSPS-G6 were obtained as well. As the results showed that, the optimal glyphosate concentration for screening transgenic cotton shoot tips was 10 mg·L^-1, the optimal agrobacterium density was 0.9-1.0 in OD₆₀₀; the infection period was about 20 min; the co-culture period with agrobacterium was 48 hours; and SH medium containing 0.5 g·L^-1 activated carbon was the best recovery medium for the transgenic shoot tip to grow. Using this transformation system, 60 glyphosate resistant plantlets were obtained from the 360 cotton shoot tips of three upland cotton cultivar or germplasms. From them, 26 plantlets were transgenic according to the PCR products, and 23 plants with the glyphosate resistant gene survived after transplanting. Based on explant numbers, the success rate of transformation in this experiment was 6.4%. The presented transformation system is an excellent method to transform the glyphosate gene, or an exogenous gene with glyphosate as a screening gene, with the advantages of high transformation rate, less chimera, and short transformation period etc.
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  Identification of Genes Related to Cotton Yield Using Affymetrix Gene Chips

  ZHANG Li-Yuan, YU Ji-Wen, WU 　Man, MA Jian-Hui, FAN Shu-Li, SONG Mei-Zhen, PANG Chao-You, LI Xing-Li, ZHANG Jin-Fa, YU Shu-Xun

  Cotton Science. 2013, 25(5): 417-425.

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  Three linesfrom high yield and low yield groups were selected from a backcross inbred line (BIL) population, which was developed from a cross between upland cotton SG747 (Gossypium hirsutum) and egyptian cotton Giza75 (Gossypium barbadense L.). Fiber RNA of 10 days post anthesis from the six BIL lines and parents lines were extracted for Affymetrix Cotton GeneChips. Through the transcriptome analysis among these lines, 1508 differentially expressed (DE) genes were identified based on a two-fold difference or greater. Clustering analysis of these DE genes showed that the high yield BIL lines and the low yield BIL lines clustered into two distinct groups. The Blast-Mapping-Annotation-KEGG analysis by Blast2GO software was performed on the DE genes, and revealed that many genes were related to plasma membrane, oxidation reduction,  response to stress and transport. 50 DE genes were selected for further analysis by RT-PCR analysis, and many fiber- and yield-related genes were identified, which would make a firm foundation for further genetic engineering and molecular breeding studies.
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  Effects of Nitrapyrin Soaking on Cotton Germination Rate and Its Salt Resistant Physiological Characteristics in Salt Stress Condition

  Tao- Rui, LIU Tao, CHU Gui-Xin

  Cotton Science. 2013, 25(5): 426-431.

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  A stimulation experiment was carried out to investigate the effect of nitrapyrin soaking of cotton seed on cotton germination rate under different salt stress conditions(0, 80, 120 mmol·L^-1 NaCl). Meanwhile, cotton seed activities of antioxidase and contents of seed water-soluble sugar, water-soluble protein, antioxidant enzymes and malondialdehyde (MDA) in cotton seed were measured during the cotton seed germination process. An obvious inhibition effect on cotton seed germination was observed under salt stress; but nitrapyrin soaking treatments significantly increased cotton seed germination rates, germination potential, and cotton seed vigor index. Compared with water soaking treatment, germination rate increased by 11.5%～58.4%, germination potential increased by 16.8%～65.0% and cotton seed vigor index increased by 42.9%～82.5% when using nitrapyrin soaking treatment at the same salt concentration level, indicating nitrapyrin soaking plays a significant role on alleviating the salt inhibition effect of cotton seed germination. Nitrapyrin soaking treatments statistically increased cotton seed enzyme activities. Compared with water soaking treatment, enzyme activities of SOD, POD, CAT increased by 57.2%～282.7%, 8.3%～139.3% and 6.4%～15.1% with nitrapyrin soaking treatments at the same salt concentration level, respectively. Furthermore, cotton seed electrolyte leakage rate and MDA content of nitrapyrin soaking were lower than that of the water soaking treatment, and the nitrapyrin soaking treatment markedly increased water soluble organic substance content in cotton seed, namely, there has a significant effect of nitrapyrin soaking on promotion of complex organic substances transformed to water soluble organic substances during the germination process. In conclusion, through 4.1×10^-3 mmol·L^-1 2-chloro-6-(trichloromethyl) pyridine soaking exert a significant effect on increasing cotton seed germination rate, promoting its salt resistant physiological characteristics under salt stress conditions.
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  Analysis on Gene Flow and Survival Competitiveness of Transferred Disease Resistance Gene Cotton and Siblings Plant(Hibiscus trionum L. and Abutilon theophrasti Medic.)

  QIU Hong-Lin, ZHANG Lin-Hua, LIU 　Chao, WU Ming-Fu, HE 　Li, ZHU Jian-Bo, WANG Ai-Ying

  Cotton Science. 2013, 25(5): 432-439.

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  The possibility of genetic drift between disease-resistant transgenic cotton, containing the chitinase and tobacco glucanase gene (pBLGC) , and siblings plant (Hibiscus trionum L. and Abutilon theophrasti Medic), was analyzed using the hybridization and DNA introgression in north of Xinjiang. And the survival competition ability of transgenic cotton and field weed was assessed through the monitor of plant height, community species and population density, biological diversity, and biomass in the cotton seedling stage, budding period, flower fluid phase, and wadding stage in the situation of bare land planting. The results revealed that distant hybridizations between disease-resistant transgenic cotton and sibling plant (Hibiscus trionum L. and Abutilon theophrasti Medic.) could not fruit, and the possibility of the target genes of disease-resistant transgenic cotton being transferred to weeds by DNA introgression is very small. Futhermore, transgenic cotton did not enhance their competitive ability with weeds populations for plant height and population density index in natural field condition with normal irrigation and bare land planting. This suggests that the possibility of the disease-resistant transgenic cotton converting into weeds is almost zero.
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  Research of Time Interval to Arthropod Community Investigation in Transgenic Cotton Field

  LUO Jun-Yu, WANG Chun-Yi, XIN Hui-Jiang, CUI Jin-Jie

  Cotton Science. 2013, 25(5): 440-445.

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  In recent years, the effects of transgenic cotton to the ecosystem of cotton field were studied mor1y, but mostly studies were concentrated on evaluating transgenic cotton biology community safety by investigating insect community diversity in cotton fields. But the results were different because of the different methods and investigating time of investigation. To understanding the effects of different investigation time to biological diversity of communities in cotton field, we investigated the arthropod animal community in transgenic cotton and conventional cotton fields in different time intervals, and compared the results of arthropod animal population, diversity index, evenness index, dominant concentration, species richness by investigating at different time points in transgenic cotton field. The results showed that these indexes were influenced the same in transgenic cotton and conventional cotton at different time points. And the total number of individuals and species richness significantly decreased with increase of the time interval, but the diversity index, evenness index and dominant concentration had not obvious change among different time intervals. A lot of occasional species and rare species of insects appeared in cotton field in recent years, so we analyzed methods for objective and accurate evaluating the arthropod community in transgenic cotton field, and further study directions.
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  Effect of Putrescine Priming on the NaCl Tolerance of Two Transgenic Insect Resistant Hybrid Cotton (Gossypium hirsutum L.)

  LIU Tian-Xiong, CHEN Jin-Hong, HE Qiu-Ling, ZHU Shui-Jin

  Cotton Science. 2013, 25(5): 446-452.

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  The effect of seed priming with putrescine on the seed germination, seedling emergence, yield， and fiber quality of the transgenic insect resistant hybrid cottons stressed by 0.5% NaCl was studied, using two extending transgenic insect resistant hybrid cotton cultivars, ZKZ-3 and ZZ-14, as the materials and extending normal cotton cultivar. The results indicated that seed priming treatment with putrescine could improve the germination percentage, germination index, mean germination time, and seedling emergence of the two transgenic hybrid cotton cultivars stressed with 0.5% NaCl significantly. On the shoal land with 0.5% NaCl, there were no significant effects of seed primer with 0.05 mmol·L^-1 putrescine on the bolls per plant, boll weight, and lint percent, but lint yield which was increased by seed primer with putrescine significantly. Also, there was no significant effect of seed primer with putrescine on the staple length, but on micronaire and fiber strength. The results of analysis for SOD, POD, and MDA in the different organs of seedlings showed that putrescine primer could increase the activities of POD and SOD in the cotton seedling plant stressed with 0.5% NaCl significantly, but decrease dramatically for the content of MDA, comparing with the checks without priming or primer with water.
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  The Influences of Wind and Bee on Pollen-mediated Gene Flow from Transgenic Cry1Ac Cotton

  HE 　Juan, ZHU Wei-Long, ZHU Jia-Lin, PAN Li-Long, ZHANG Qing-Wen, LIU Xiao-Xia

  Cotton Science. 2013, 25(5): 453-458.

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  The frequency and distance of pollen-mediated gene flow from transgenic cotton to non-transgenic cotton were investigated in greenhouses using artificial wind, or bee pollinators, separately. The seedlings of F₁ generations were screened using PCR assays, then the positive samples were identified for Bt insecticidal protein by dipstick assay. The results showed that the frequency of gene flow in the greenhouse with wind or bee pollinators was higher than that of the control (without wind or bee pollinators). The pollen-mediated gene flow (PGF) to the non-transgenic counterpart (Shiyuan 321) was obviously higher than those to the island cotton (GIZA 1) and another upland cotton (CCRI 35) in both wind and bee pollination treatments. Furthermore, in both treatments, there was a remarkable difference in the PGF to Shiyuan 321 related to distances from the donor plots; whereas this trend was not obvious to the other two conventional cultivars. There were a total of 72 positive samples in the wind treatment. The maximal distance of gene flow to Shiyuan 321 was 25.6 m, while the maximal distance to both CCRI 35 and GIZA 1 was 19.2 m. In the bee treatments, there were a total of 75 positive samples. The maximal distances of gene flow to all three conventional cultivars was 36 m, and gene flow peaked at this distance. Our research builds a foundation for the model of gene flow, and provides value reference for the ecological risk assessment of transgenic cotton.
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  Monitoring of Cry1A Expression and Efficacy to Cotton Bollworm in Bt Cotton Varieties Planted in Hebei, Shandong and Henan Provinces

  吕Li-Min , LUO Jun-Yu, LIU Quan-Yi, YANG Zi-Shan, ZHANG 　Shuai, WANG Chun-Yi, CUI Jin-Jie

  Cotton Science. 2013, 25(5): 459-466.

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  From 2008 to 2012, using the methods of field monitoring, bioassay, expression of Bt exogenous proteins, we detected 129 Bt cotton varieties at 44 plant cotton sections in Hebei, Shandong and Henan cotton region. The field monitoring revealed that No. of remnant cotton bollworm per 100 plants was 20 for the second generation cotton bollworm in 2011, over the control index, but that of other years lower than the control index;  the rate of injured cotton stem-tips, buds and bolls were at a low level. The results of the bioassay showed that the corrected mortality for the second generation bollworm increased at first but then decreased, and that of the third and fourth generation bollworm showed a decreasing tendency. The Bt exogenous protein assays showed expression was decreasing year by year at the seedling stage, whereas the expression of Bt exogenous protein in the budding and bolling stage were increasing a little, but lower than those in seedling stage.
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  A Protocol for Total RNA Isolation Suitable for Real-time PCR Analysis of Cotton MicroRNAs

  YU Dao-Qian, WANG Gaskin, DU Xiong-Ming

  Cotton Science. 2013, 25(5): 467-470.

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  A method for isolation of total RNA species is presented. The modified guanidinium isothiocyanate- ammonium thiocyanate-acid phenol method was used to extract total RNA from fiber and ovule of cotton(Gossypium hirsutum L.). The stem-loop Real-time PCR method was selected to identify mature miRNA. Experiments validated that the method we used was suitable for quantitative analysis of miRNA because of the high quality of total RNA and high recycling efficiency of miRNA.