Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.

Insect Resistance of Different Tissues of Transgenic Cotton to Spodoptera exigua(Hbner)

[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.

Effect of Transgenic Bt plus CpTI Cotton on Carboxylesterase and Acetylcholinesterase of Cotton Aphid Aphis gossypii

[Objective] The research aimed to assess the effect of transgenic Bt plus CpTI cotton variety SGK321 on carboxylesterase and acetylcholinesterase of cotton aphid Aphis gossypii and provide theoretical basis for studying the biosafety of transgenic cotton.[Method] Cotton aphids were fed with SGK321 and Shiyuan321（normal parental varieties） for over 40 generations.Enzyme activities were compared between cotton aphids feeding on SGK321 for 1,2,3,41,42 and 43 generations with those on Shiyuan321.[Result] The carboxylesterase activity of cotton aphids feeding on SGK321 for 1 generation was significantly higher than those feeding on Shiyuan321.Acetylcholinesterase activity of cotton aphids feeding on SGK321 for 1,2 and 3 generations were significantly higher than those feeding on Shiyuan321 in the same generation.But there was no significant difference of enzyme activity between cotton aphids feeding on SGK321 for a long term and those feeding on parental cotton.[Conclusion] The cotton aphid that feeding on transgenic Bt plus CpTI cotton SGK321 for a long time has adaptivity to SGK321 by regulating the detoxifying enzyme.

Obtaining High Pest_resistant Transgenic Upland Cotton Cultivars Carrying cry1Ac3 Gene Driven by Chimeric OM Promoter

Hypocotyl segments from aseptic seedlings of two important cultivars of upland cotton ( Gossypium hirsutum L.) in Northwest China, 'Xinluzao_1', 'Jinmian_7', 'Jinmian_12' and 'Jihe_321' were transformed respectively by two efficient plant expression plasmids pBinMoBc and pBinoBc via Agrobacterium tumefaciens . In pBinMoBc, cry 1Ac3 gene, which encodes the Bt toxin, is under the control of chimeric OM promoter. In pBinoBc, it is under control of CaMV 35S promoter. After co_cultivation with Agrobacterium tumefimpfaciens LBA4404 (containing pBinMoBc or pBinoBc), kanamycin_resistant selection, somatic embryos were induced and regenerated plants were obtained. Then the regenerated plantlets were grafted to untransformed stocks in greenhouse to produce descendants. The integration of cry 1Ac3 gene and its expression in T 2 generation of transgenic cotton plants were confirmed by Southern hybridization and Western blotting. The analyses of insect bioassay indicated that the transgenic plants of both constructions have significant resistance to the larvae of cotton bollworm ( Heliothis armigera ) and that cry 1Ac3 gene driven by chimeric OM promoter could endue T 2 generation cotton with high pest_resistant ability, implicating that it has a profound application in genetic engineering to breed new pest_resistant cotton varieties.

Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

[Objective] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Method] Btcry1Ac toxic protein expression in roots, stems and leaves of transgenic cotton Guoshen GK45 at different developmental stages and the annual average content of BtCry1Ac toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCry1Ac toxin protein decreased during the growth process of insect-resistant transgenic cotton; to be specific, the content of BtCry1Ac toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCry1Ac toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCry1Ac toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of Btcry1Ac toxic protein provides scientific basis for the risk assessment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

Breeding of Transgenic Hybrid Cotton Variety‘Yumian 50' and Cultivation Technology

The mid-early-maturing new cotton variety ＇Yumian 50＇ was bred by In-stitute of Industrial Crops, Henan Academy of Agricultural Sciences. It was exam-ined and approved by Henan Provincial Crop Variety Approval Committee in 2016 （Yushenmian 2016002）. In order to study the variety characteristics of ＇Yumian 50＇ and the key points of its cultivation technology and accelerate its application in cot-ton production, the breeding process of ＇Yumian 50＇ was described in this paper. The test data of 26 times for 3 years of ＇Yumian 50＇ participating in Henan region-al tests in 2012-2014 were summarized. The results showed that ＇Yumian 50＇ had outstanding performance in Henan regional tests and production tests. ＇Yumian 50＇ is a new cotton variety with characteristics of high yield, good quality and high re-sistance to diseases and pests.

Impact of Transgenic Bt+CpTI Cotton on Soil Enzyme Activities and Soil Microorganisms

Due to its strong and effective insecticidal properties, transgenic Bt＋CpTI cotton has witnessed an expanding planting area in recent years, and the impact of its cultivation on soil ecosystem becomes an important part of environmental risk assessment. Using transgenic Bt＋CpTI cotton sGK321 and its parental homologous conventional cotton Shiyuan 321 as the study objects, a comparative analysis was conducted on the changes in enzyme activities （urease, alkaline phosphatase, and catalase） of the rhizosphere soil and changes in the number of culturable microor-ganisms （bacteria, fungi, and actinomycetes） at different growth stages （seedling stage, budding stage, flower and bol stage, and bol opening stage） of sGK321 and Shiyuan 321 under the condition of 13 years field plantings. The results showed that, the populations of bacteria, fungi, and actinomycete and the soil enzyme activi-ties of urease, alkaline phosphatase and catalase had a similar variation trend along with the cotton growing process for transgenic cotton and conventional cotton. Some occasional and inconsistent effects on soil enzyme activities and soil fungi composi-tion in the rhizosphere soil of transgenic Bt＋CpTI cotton were found at the seedling stage, budding stage, flower and bol stage as compared with that of conventional cotton. The amount of bacteria and actinomycetes were not significantly different during a certain stage; however, the activities of urease, catalase, alkaline phos-phatase, also with the number of fungi were significantly different, e.g. the urease activities at seedling stage, the alkaline phosphatase at seedling and budding stages, and the soil culturable fungi at flower and bol stage were less than that of conven-tional cotton, while the soil alkaline phosphatase activities at flower and bol stage were higher. Cluster analysis showed that soil enzyme activities and microbial popu-lation changed mainly along the growth processes, suffering little from the planting of transgenic Bt＋CpTI cotton.

Effects of Transgenic Bt+CpTI Cotton on Field Abundance of Non-Target Pests and Predators in Xinjiang, China

Transgenic insect-resistant cotton is being increasingly planted in Xinjiang cotton-planting regions, where geographical climate conditions and species composition of pests and natural enemies are greatly unique in China. Limited studies have been conducted on the ecological impacts of transgenic insect-resistant cotton, especially for transgenic double genes （Bt＋CpTI） cotton, in this region. In this study, the potential effects of transgenic Bt＋CpTI cotton on the seasonal abundance of non-target pests and predators were assessed from 2009 to 2011 in Korla, Xinjiang. The results showed that species composition and seasonal abundance of 5 groups of pests and 5 groups of predators were not significantly different between transgenic Bt＋CpTI cotton and non-transgenic cotton every year. It suggests that transgenic Bt＋CpTI cotton per se does not affect the population dynamics of non-target pests and predators on this crop in Xinjiang.

NaCl-Induced Changes of Ion Fluxes in Roots of Transgenic Bacillus thuringiensis (Bt) Cotton (Gossypium hirsutum L.)

Bacillus thuringiensis （Bt） cotton is grown worldwide, including in saline soils, but the effect of salinity on ion fluxes of Bt cotton remains unknown. Responses of two transgenic Bt cotton genotypes （SGK321 and 29317） and their corresponding receptors, Shiyuan 321 （SY321） and Jihe 321 （J321）, to 150 mmol L-1 NaCl stress were studied in a growth chamber. The root dry weight of SGK321 and 29317 under NaCl treatment was decreased by 30 and 31%, respectively. However, their corresponding receptor cultivars SY321 and J321 were less affected （19 and 24%, respectively）. The root length and surface area of the Bt cultivars were significantly decreased relative to their receptors under salt stress. NaCl treatment significantly increased CrylAc mRNA transcript levels in SGK321 and 29317 but did not affect Bt protein content in leaves or roots of either cultivar at 1 and 7 d after NaCl treatment. Fluxes of Na^＋, K^＋, and H^＋ in roots were investigated using the scanning ion-selective electrode technique. Both mean K^＋ efflux rate and transient K^＋ efflux of the Bt cultivars increased four-fold compared to their corresponding receptors when exposed to salinity stress. There were no significant differences in Na^＋ efflux between Bt and non-Bt cottons. Furthermore, the Na^＋ contents in roots and leaves of all genotypes dramatically increased under salt stress, whereas K^＋ contents decreased. Our results suggested that Bt cotton cultivars are more sensitive to salt stress than their receptor genotypes.

Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos

The 5＇ fragment （1 647 bp） of the cotton glucuronosyltransferase gene （GhGlcAT1） was transcriptionally fused to the β-glucuronidase （GUS） gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars （glucose and sucrose） as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from -281 to ＋30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element（s） for sucrose induction might be located between -635 and -281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugarresponsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGIcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGIcAT 1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.

Overexpression of AmDUF1517 enhanced tolerance to salinity, drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmD UF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmD UF1517 cotton displayed significantly higher antioxidant enzyme（superoxide dismutase（SOD）, peroxidase（POD）, catalase（CAT）, and glutathione S-transferase（GST）） activity and less reactive oxygen species（ROS） accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.

Combining Ability and Heterosis Between High Strength Lines and Transgenic Bt (Bacillus thuringiensis) Bollworm-Resistant Lines in Upland Cotton (Gossypium hirsutum L.)

To analyse the combining ability and heterosis between high-strength lines and transgenic Bt bollworm-resistant lines in upland cotton, 5 high-strength lines were crossed as female lines with 12 transgenic Bt bollworm-resistant lines according to NCII design. It was demonstrated that the compositions of variance in various traits were quite different. For seed cotton yield, lint yield, boll numbers per plant and boll weight, the dominant (special combining ability) effects were the major effects, accounting for 87.38, 84. 40, 80. 04 and 64. 46% of the total phenotypic variances, respectively, while for fibre strength and micronaire value, the additive (general combining ability) effects had the major effects, with a ratio of additive variance to phenotypic variance of 78.85 and 43.80%. As for lint percent and 2.5% span length, the dominant and additive variances had similar effects, in phenotypic variances (54.94 and 40.11% for lint percent, 45.76 and 42.49% for 2. 5% span length, respectively). The mid-parent heterosis (Hpm), surpassing parent heterosis (Hpb) and competitive heterosis (Hck) for seed cotton yield and lint yield were both extremely significant. For fibre properties, the Hck and Hpm of 2.5 % fibre span length were extremely significant, the Hck of fibre strength was significant, and the favorable negative Hck of micronaire was also extremely significant. The increments of hybrid over common variety were 17% for lint yield and fibre strength, 7% for fibre span length, and 4 % for fineness.

Development of Transgenic Restorer of Cytoplasmic Male Sterility in Upland Cotton

A glutathione S-transferase gene (gst) has been introduced into restorers of cytoplasmic male sterility in upland cotton (Gossypium hirsutum L.) using Agrobacterium-mediated transformation. A trans-genie restorer, signed as 'Zheda strong restorer', which has strong restorability to male sterility, was selected from progeny plants of transformants. When compared with an American restorer 'DES-HAF277', the fertility restorability of 'Zheda strong restorer' to male sterility was been enhanced by 25.8% in the percentage of viable pollens of hybrid (sterile line × restorer) F1 The hybrid gave 3.6 more bolls per plant, 10.1% less aborted seeds and 10.6% more lint yield when 'Zheda strong restorer' was used as male parent than when 'DES-HAF277' was. Southern and Northern bloting analysis showed that the foreign gsf gene was detectable and highly expressed in 'Zheda strong restorer'.

Effects of Transgenic Bt+CpTI Cotton Cultivation on Functional Diversity of Microbial Communities in Rhizosphere Soils

[Objective] This study aimed to investigation the effects of tranagenic Bt ＋ CpTI cotton cultivation on functional diversity of microbial communities in rhizospbere soils. E Method] By using the Biolog method, a comparative study was conducted on the utilization level of single carbon source by microbes in the rhi- zosphere soils of transgenic Bt ＋ CpTI cotton sGK321 and its parental conventional cotton ＇ Shiyuan 321＇ at different growth stages. [ Result ] The results showed that, compared with the parental conventional cotton, the average well-color development （AWCD） value of micmhial communities in rhizospbere soils of transgenie Bt ＋ CpTI cotton were significantly higher （P 〈 O. 05） at seedling stage and budding stage while significantly lower at flower and boll stage and bell opening stage. Shannon-Wiener diversity index （H） and Simpson dominance index （D） of microbial communities in rhlzesphere soils of transgenic cotton and conventional cotton varied with the different growth stages, whereas the Shannon-Wiener evenness index （E） showed no significant difference between transgenie cotton and convention- al cotton at four growth stages. Principal component analysis indicated that the patterns of carbon source utilization by microbial communities in rhizospbere soils were similar among transgenic cotton at seeding stage and flower and boll stage and parental conventional cotton at seeding stage and budding stage, which were also similar between tranagenic cotton at budding stage and parental conventional cotton at flower and boll stage. [ Conclusion] Analysis of different carbon sources indi- cated that the main carbon sources utilized by soil microbes were carbohydrates, amino acids, carboxylie acids and polymers.

Evaluation of Impact of Pollen Grains from Bt,Bt/CpTITransgenic Cotton and Bt Corn Plants on the Growth andDevelopment of the Mulberry Silkworm,Bombyx moriLinnaeus (Lepidoptera:Bombycidae)

The S-endotoxin genes of Bacillus thuringiensis (Bt) and proteinase inhibitor (PI) genes are two kinds of genes popularly used for developing transgenic plants resistant to insect pests. To clarify whether there is any risk concerning the effects of pollens from these transgenic crops on non-target insects with economic importance, such as the effects on the growth and development as well as cocoon quality of the silkworm, Bombyx mori Linnaeus, a series of feeding experiments were conducted, using pollens from transgenic cotton or corn containing cry 1Ac, cry1A+CpTI or crylAb genes, compared with pollens from non-transgenic normal cotton and corn as well as the non-pollen treatment. In contrast to the latter ones, pollens from transgenic plants showed no significant adverse effects on larval mortality, cocoon weight, pupa weight, cocoon shell weight, pupation rate, emergence rate and fecundity of the silkworm after neonates were fed with the pollens for 72 h. In addition, no dosage effects of pollens were found. Though the duration of 1st instar larvae was prolonged in the case of feeding with transgenic pollens as compared with those of the non-pollen treatment , but they were not significantly different from those fed with pollens from non-transgenic cotton or corn. Meanwhile, the body weight of the 3rd instar molters fed with transgenic pollens was obviously different from those for non-pollen treatment, and was all significantly heavier than that of the controls. Consequently, it is considered that the adverse effect of pollens from transgenic insect-resistant cotton and corn on the growth and development of the silkworm is negligible.

Development of Transgenic Cotton Resistant to Fungal Diseases and of Mutants by T-DNA Insertional Mutagenesis

Verticillium(V.dahliae Kleb.) and Fusarium(F.Oxysporum f.sp.vasinfectum)wilt aretwo major fungal diseases in cotton productionwhich cause great crop damage and yield lossworld wide.Breeding and application ofresistant varieties have effectively controlledFusarium wilt.However,the Verticillium wilt-

Study on the Handle of Keratin Transgenic Cotton Fabric

Gene of animal keratin can be inoculated into cotton fiber and thus get the keratin transgenic cotton fiber through transgenic technology. Handle of two kinds of pure cotton poplin, one of which is made of the keratin transgenic cotton while the other is made of the ordinary cotton of the same breed as control group and both with absolutely identical spinning, weaving, and dyeing process, was objectively evaluated with KES system. The result of analysis indicates that the principal changes of keratin transgenic cotton fabric are that the bending and shearing property of the fabric are considerably enhanced, KOSHI (Stiffness) and HARI (Anti-drape stiffness) of the fabric are good, while SHINAYAKASA (Flexibility with soft feeling) and SHARI (Crispness) decline.

Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

An insect excitatory toxin gene from Buthus martensii Karseh （BmKIT） was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Eseherichia coli BL21 （DE3） host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyelonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.

Dissection of Genetic Effects of Quantitative Trait Loci(QTL) in Transgenic Cotton

When alien DNA inserts into cotton genome in multi-copy manner,several QTL in cotton genome are disrupted,which are called dQTL in this study.Transgenic mutant line is near-isogenic to its recipient which is divergent for the dQTL from remaining QTL.So,a set of data from a