Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids

To evaluate the possible antiaphid function of Amaranthus caudatus agglutinin (ACA) in allogenetic plants, transgenic cotton plants expressing the ACA gene under the control of a phloem‐specific promoter were generated via Agrobacterium‐mediated gene transformation. Based on the results of Southern blot analyses, six plants with single or lower copy transgene and favourable agronomic traits were selected for further studies. ACA expression levels ranged from 0.02% to 0.45% of total soluble protein as determined by Western blot analysis in the six selected transgenic plants. Insect bioassays using nymphs of cotton aphid (Aphis gossypii Glover) showed that five of the six transgenic plants significantly inhibited the population growth of cotton aphid, with the highest inhibition rate of 64.5%. These results shed some new light on the antiaphid function of the ACA gene as well as the promising application of the gene for obtaining aphid‐resistant transgenic cotton plants to reduce the yield loss and honeydew contamination of fibre by aphids.     

The Diagnostic Model of Cotton Aphids Based on Leaf Textural Features

[Objective] The aim of this research is to explore the possibility of rapid identification of aphid damage to cotton leaves by identifying the hyperspectral image about healthy and aphid damage cotton leaves. [Method] Taking Xinluzao 45 as material, hyperspectral images of healthy and damaged cotton leaves by aphids were obtained, and hyperspectral image information was extracted from interested regions of different cotton samples. Then three descending dimension methods were used to extract hyperspectral feature, and Gray-Level Co-occurrence Matrix to extract image texture feature. Finally a cotton aphids damage diagnostic model was built up. [Result] The prediction accuracy based on all textural samples input Random frog-partial least-square-linear discriminant Function (RF-PLS-LDA) model was 91.49%. The prediction accuracy based on energy data input principal component analysis-loading partial least-square-linear discriminant function (PCA-Loading-PLS-LDA) model was 92.55%. [Conclusion] The second-order statistics (energy) of gray co-occurrence matrix can be used to simplify the model, reduce the computation and improve the stability of prediction. Based on the texture feature vector, the identification of aphid cotton leaves can be realized effectively, which provides a method for the rapid identification of insect pests.     

棉花–小麦条带种植对棉花苗蚜发生及为害的影响

为了探究棉花–小麦条带种植对棉花苗蚜发生及为害的影响,对棉花苗期的蚜虫种群数量及为害情况进行了调查分析。结果表明,棉花–小麦条带种植模式可减轻棉花苗期蚜虫的发生和为害,苗期有翅蚜和无翅蚜数量均显著低于常规种植,分别减少70.4%和84.8%,有蚜株率和卷叶株率分别较常规种植降低67.4%和81.3%;棉花条带中,边行对棉蚜的控制作用显著优于中间行,边行有翅蚜和无翅蚜数量分别比中间行减少了43.7%和54.3%,有蚜株率和卷叶株率分别较中间行降低了52.4%和84.6%。     

农杆菌介导的RNAi CP基因在大豆中的转化

花叶病毒(soybean mosaic virus, SMV)病是大豆主要病害之一,生产上常采用种植抗性品种方法来防治。本研究以RNA干扰花叶病毒衣壳蛋白(coat protein, CP)基因为表达载体,Bar基因作为筛选标记基因,成熟子叶节为外植体,采用农杆菌介导法获得了22株T₀代转基因大豆生根苗,经草丁膦涂抹、Bar试纸条和PCR法鉴定,获得RNAi CP转基因植株18株;对转基因植株T₁代的遗传分析表明,外源基因能够稳定遗传到下一代且符合孟德尔遗传规律;T₁代Southern杂交表明,导入的干扰片段为单拷贝;花叶病毒摩擦接种表明RNAi CP转基因大豆植株具有抗花叶病毒特性;摩擦接种后3周,DAS-ELISA检测进一步表明,RNAi CP转基因植株花叶病毒检出率仅为7.69%,而非转基因植株为100%。这表明RNAi花叶病毒CP基因可用于抗大豆花叶病毒的研究。     

大豆花粉管通道技术转化雪花莲凝集素(GNA)基因

采用花粉管通道技术,用雪花莲凝集素基因（Galanthus nivalis agglutinin,GNA）转化吉林省主推品种吉林20号、吉林30号、吉林45号品种大豆。通过接蚜鉴定和PCR鉴定,从所获得的种子苗中筛选出转基因植株。对转基因植株的后代进行分子生物学鉴定：（1）PCR分析,转基因植株97TGR1和97TGR2的T2代表现阳性,第5代表现阳性纯合;97TGR1、97TGR2和98FD1～98FD20的T3代Western blotting检测结果证明,GNA基因在蛋白质水平有表达,最高表达量占总可溶性蛋白的0．7％;97TGR1、98TGR2和99JI45 TGR2的Southern blotting检测结果显示,GNA基因已插入大豆基因组;（2）遗传学分析,97TRG1的T2代呈孟德尔3：1分离,97TGR2的T3代出现种皮颜色不规则分离。经过抗蚜性鉴定和连续的筛选,获得抗性纯系;（3）抗蚜性鉴定,转基因株的T1、T2世代转基因植株可抑制蚜虫繁殖量50％～90％;（4）品系鉴定,转基因大豆的抗蚜性达到农学标准抗（R）和高抗（HR）水平;大面积环境释放试验自然感蚜鉴定,转基因系蚜虫发生的高峰比对照延迟,高峰期过后群体蚜量的下降速度也比对照快。本研究认为,大豆花粉管通道技术可以利用于大豆的转基因研究和应用中,GNA基因在改良大豆的抗蚜性上是可取的。     

用基因枪法获得转异天南星基因aha抗蚜虫小麦

凝集素是一类具有特异糖结合活性的蛋白,对蚜虫等害虫有很强的抗杀作用。利用异天南星凝集素基因aha(Arisaema heterophyllum agglutinin)以及水稻Rubisco小亚基启动子,构建了aha基因植物绿色组织特异性表达载体,并采用基因枪转化方法, 与携带bar基因的pAHC20载体共转化到小麦品种科农199中。经过愈伤诱导、再生和筛选以及PCR鉴定,获得aha转基因植株42株,平均转化效率为2.41%。对转aha基因植株后代PCR鉴定表明,T₁代转基因植株的分离比例基本符合孟德尔遗传规律。利用室内多目标综合判别法评定抗蚜虫特性,8个T₁代转基因株系中有高抗材料1份,低抗材料3份,占参试比例44.4%。本研究为获得抗蚜虫转基因小麦新材料奠定了基础。     

中国北方棉区转基因抗虫棉对棉苗蚜及其两种天敌的影响(英文)

2 0 0 1年田间小区试验研究了转 Bt基因棉和转双价基因 ( Bt+ Cp TI)棉对棉苗蚜及其天敌种群动态的影响 ;室内研究了转基因抗虫棉对两种棉蚜天敌的生物学影响。结果表明 ,转基因抗虫棉对棉蚜种群数量的影响不明显 ,未达显著水平。单作转基因棉田棉蚜发生期比麦套棉田提前 5天左右 ,发生数量是麦套棉田的 1 .6倍 ,表明麦棉套作可有效的控制苗蚜的为害。室内研究表明 ,异色瓢虫对用 Bt棉处理的的棉蚜的捕食量比对照增加 2 2 .0 % ,棉蚜茧蜂对用 Bt棉处理的棉蚜的寄生率降低 2 3.5 %。     

中国北方棉区转基因抗虫棉对棉苗蚜及其两种天敌的影响（英）

2001年田间小区试验研究了转Bt基因棉和转双价基因(Bt+CpTI)棉对棉苗蚜及其天敌种群动态的影响;室内研究了转基因抗虫棉对两种棉蚜天敌的生物学影响.结果表明,转基因抗虫棉对棉蚜种群数量的影响不明显,未达显著水平.单作转基因棉田棉蚜发生期比麦套棉田提前5天左右,发生数量是麦套棉田的1.6倍,表明麦棉套作可有效的控制苗蚜的为害.室内研究表明,异色瓢虫对用Bt棉处理的的棉蚜的捕食量比对照增加22.0%,棉蚜茧蜂对用Bt棉处理的棉蚜的寄主率降低23.5%.     

Construction of RNAi Vector of Cotton (Gossypium hirsutum L.) FAD2-1 Gene and Acquisition of New High Oleic Acid Germplasm

[Objective] Here, the aim was to create a new cotton (Gossypium hirsutum L.) germplasm with high oleic acid and low linoleic acid contents without affecting the fiber yield and quality. [Method] The fatty acid desaturation 2-1 (FAD2-1) gene catalyzes the formation of polyunsaturated linoleic acid from monounsaturated fatty acid in cotton. The protein’s properties, structure and functions were analyzed using the information analysis method. A conserved 381-bp fragment of GhFAD2-1 was cloned to construct an RNA interference vector, which was transformed into cotton by Agrobacterium-mediated hypocotyl infection. The fatty acid compositions and contents of T₂―T₄ transgenic plants were determined using gas chromatography-mass spectrometry. The T₄ transgenic lines were used to investigate the copy number, target gene expression, agronomic traits, and fiber quality. [Results] The RNA interference vector of GhFAD2-1 was successfully constructed and transformed into cotton. The target gene’s expression level was significantly lower in transgenic plants than in controls. The transgenic plants had high oleic acid and low linoleic acid contents, which could be stably inherited by the offspring. The oleic acid contents of transgenic cotton seeds increased by 224.1%, and the linoleic acid content decreased by 237.5%, compared with the control seeds. There were no significant differences in the agronomic traits and fiber quality of the transgenic lines compared with the control. [Conclusion] These results verified the function of GhFAD2-1 in cotton and provide a basis for breeding cotton varieties with high oleic acid contents.     

转人工合成中国水仙凝集素基因(sNTL)小麦的获得及其抗麦长管蚜效果分析

为了丰富可利用的植物凝集素基因,获得具有较高抗蚜水平的小麦新种质,采用"Gene Shuffling"策略人工合成中国水仙凝集素基因(s NTL),并转化小麦以分析其抗蚜效果。结果表明:根据小麦遗传密码偏好性人工合成了植物凝集素s NTL基因,并构建了s NTL表达载体;用基因枪转化小麦品种中优9507和丰优6号的幼胚愈伤组织,经PPT筛选和分化后,获得T0植株,对T0植株进行PCR筛选而获得阳性植株28株。并经过连续PCR检测,表明s NTL基因已经初步与小麦基因组进行整合并且能够稳定表达。对T2转基因株系进行室内离体叶片抗虫效果分析,结果表明,转s NTL株系使蚜虫致死率达到53.1%~59.4%,其中转基因株系zy9-4的蚜虫致死率最高,比其对照品种中优9507高24.8%,同时其蚜虫增长率最小为22.1%,比对照品种中优9507低了约49.6%;转基因株系fy4-2使蚜虫致死率比对照(丰优6号)高约14.1%,而其蚜虫增长率比对照降低达到33.4%。对T3转基因株系田间鉴定结果显示,同对照中优9507相比,zy9-4、zy9-8单分蘖蚜虫发生量分别降低51.4%,45.1%,达到显著或极显著水平;fy4-2、fy4-4与对照丰优6号相比,单分蘖蚜虫发生量降低水平分别为58.7%,76.9%。综合室内与田间抗虫鉴定结果证明,利用人工合成的s NTL基因可以有效地提高小麦的抗蚜性。     

转基因抗虫棉对棉蚜生长发育和繁殖的影响

以常规棉泗棉3号上饲养10代的棉蚜作为虫源,以其亲本为对照,在室内分别研究了转单、双价基因抗虫棉对棉蚜的短期影响,及单价抗虫棉对棉蚜的长期效应。结果发现,单价抗虫棉GK12和双价抗虫棉SGK321在短期内对棉蚜生长发育、繁殖和生命表参数无显著影响。在抗虫棉上取食1代的棉蚜,各龄发育历期、成虫寿命、产仔数和产仔历期、内禀增长率、净增长率、平均发育历期等和取食亲本的棉蚜之间均无显著差异。在GK12上取食37代的棉蚜与取食亲本常规棉泗棉3号和在GK12上取食1代的棉蚜之间以上各指标也无显著差异。可见,单价抗虫棉GK12和双价抗虫棉SGK321在短期内对棉蚜的生长发育和繁殖均无显著影响,GK12在长期内对棉蚜也未产生显著影响。     

Generation and Glyphosate Resistance Analysis of G10eve Transgenic Cotton

[Objective] Novel germplasm of cotton was generated for development of cultivars with high resistance to glyphosate. [Method] The glyphosate resistance gene G10eve was preliminarily analyzed using bioinformatics methods and introduced into 'Coker 312' via Agrobacterium-mediated transformation. The expression of G10eve was estimated by PCR (Polymerase chain reaction) and qRT-PCR (Quantitative real-time PCR). The content of shikimic acid and tolerance to glyphosate were determined in response to application of glyphosate to 'Coker 312' and transgenic plants. [Result] A total of 28 independent positive transgenic lines were obtained. The transformation efficiency and the rate of seedling differentiation were up to 49.3% and 40.6%, respectively. PCR and qRT-PCR analysis showed that G10eve was integrated into the cotton genome, and the G10eve expression level differed significantly among the transgenic lines. At the cotyledon stage, the transgenic cotton showed resistance to 8 mL·L^－1 glyphosate, whereas 'Coker 312' showed phytotoxicity to the 2 mL·L^－1 glyphosate treatment. No significant accumulation of shikimic acid was detected in transgenic plants before or after glyphosate treatment, which indicated the typical physical characteristic of glyphosate resistance. [Conclusion] Overexpression of G10eve in cotton improved resistance to glyphosate. Thus, novel germplasm of cotton resistant to glyphosate was generated.     

The Difference and Mechanism in Tolerance to Low-Potassium among Cotton Seedlings Over-Expressed Arabidopsis AtCIPK 23, AtAKT 1 and AtCBL 1 Genes

[Objective] To provide theoretical basis for potassium efficient cotton breeding, this work aimed to evaluate the influence of low potassium on transgenic cotton seedlings over-expressing Arabidopsis thaliana’s AtCIPK 23, AtAKT 1 and AtCBL 1 genes, respectively. [Method] Hydroponics was used to explore the growth vigor, dry weight, potassium concentration and accumulation of transgenic cotton and CCRI 24 seedlings under low and optimum potassium conditions. Furthermore, the root morphology, potassium utilization index, potassium transport ability and the severity to damage were comprehensively analyzed to uncover the possible physiological mechanisms. [Result] Under the optimum conditions of potassium, the transgenic plants and control grew well and showed no significant difference. However, with low potassium stress, the dry weight of plants over-expressing AtAKT 1 gene was 1.66 times of CCRI 24. Its total root length, root surface area and volume were about 3 times than those of CCRI 24. The AtAKT 1 plants also displayed obvious growth advantage, higher potassium utilization index and transport ability, and the severity to damage was lower. Plants over-expressing AtCIPK 23 and AtCBL 1 genes, however, were almost the same as the CCRI 24. [Conclusion] This study revealed that the ability of cotton’s resistance to low potassium was significantly improved by over-expression of AtAKT 1 gene, an exogenous potassium ion channel protein gene from Arabidopsis thaliana.     

安阳棉蚜寄主专化型及其形成机制初步分析

为了解安阳地区棉蚜寄主专化型情况,进行了棉蚜不同寄主转接的适合度测定。结果显示,棉花上与黄瓜上的棉蚜相互转接后其存活率及繁殖力均明显下降,不能建立种群;棉花上的棉蚜转接到西葫芦上时,其存活率及繁殖力与其原寄主无显著差异;黄瓜上的棉蚜转接到西葫芦上时,其净增殖率及内禀增长率均显著地高于其原寄主;黄瓜上棉蚜对西葫芦的适应性比棉花上棉蚜强。表明安阳地区棉蚜存在黄瓜和棉花两种寄主专化型,且都可以利用西葫芦。进一步对3种植物苗期叶片游离氨基酸进行测定,结果显示,游离氨基酸种类及总量均在西葫芦中最多,棉花中次之,黄瓜中最少。寄主植物游离氨基酸种类及含量的多少会影响棉蚜的生长与繁殖,可能是棉蚜寄主专化型形成的影响因素之一。     

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.     

高通量测序技术研究棉蚜携带病毒种类

棉蚜是传播寄主植物病毒的主要昆虫媒介,但对棉蚜携带的病毒种类及病毒基因组序列信息了解较少。本文利用高通量测序技术,对棉蚜田间自然种群所携带的病毒基因序列进行发掘、鉴定和分类。通过对田间苗蚜和伏蚜转录组数据进行分析,获得18种病毒基因组序列,其中17种病毒为未报道过基因组信息的种类。在18种病毒中,有13种为正链RNA病毒。苗蚜携带病毒的种类和数量都远远高于伏蚜,苗蚜携带全部的18种病毒;伏蚜仅携带8种,而且携带病毒的FPKM(Fragments per kb per million fragments)值较低。本研究丰富了棉蚜所携带的病毒基因组信息,为棉蚜传毒机制研究打下基础。     

Testing for pea leafroll virus and inheritance of resistance in peas

Summary Different aspects of pea leafroll, the most important virus disease of peas and Vicia-beans in The Netherlands, are discussed. These aspects include the spread of the virus, the damage done by the disease, the symptoms, the nature of the virus, the vectors and the host plants. Pea leafroll virus (PLRV) is a persistent virus which is invariably transmitted by aphids. The most important vector is the pea aphid, Acyrthosiphon pisum Harris. Lucerne actas as a winter host for the virus and the pea aphid. The pea aphids are reared on diseased lucerne plants and are used in a glass-house experiment to test cross populations for resistance. In view of its importance for that test it is necessary to discuss the biology of the pea aphid in some detail. Besides the glasshouse test some field test methods are mentioned.Crosses between Cobri (resistant) and Gloire de Quimper (susceptible) as well as between Wyola (resistant) and Joserva (susceptible) served to study the resistance to the virus in peas. The results of testing well over 3600 plants justified the conclusion that the resistance is inherited as a single recessive character. It is suggested to refer to the gene for susceptibility to PLRV as Lr.     

Regulation of Free Amino Acid in Cotton at the Seedling Stage by Aphis gossypii Feeding

[Objective] Free amino acids are important nitrogen nutrients, and their changes reflect the interaction between insects and host plants. The purpose of this experiment was to study the effect of aphid feeding on the free amino acid content of host cotton in seedling stage. [Method] The seedling cotton infected by aphid with different damage grades was selected from the field and analyzed the content of free amino acid in euphylla, cotyledon, hypocotyl and root. [Result] The content of free amino acids in cotton tissues increased after feeding by Aphis gossypii, and the content of free amino acids in cotyledon, hypocotyl and root is more than 2 times of the level 0. The content of non-essential amino acids increased more significantly than that of essential amino acids. The changes of single free amino acid content in different tissues were not completely consistent, and the Asn and Glu increased significantly in all tissues. [Conclusion] The feeding behavior of cotton aphid affects the nutritional level of cotton tissue and the content of free amino acid increased significantly.     

高衣分转Bt+GNA双价基因抗虫棉新种质的选育与利用

本研究对转觑和GNA双价基因后代植株,经南繁加代、抗虫检测、自交、单株和株系选择、纤维品质测定和产量比较试验,选育出双价转基因抗虫棉种质系BGsm16。该种质系高抗棉铃虫等鳞翅目害虫,对蚜虫有一定的抑制作用,抗虫性稳定且不随世代的增加而减弱,衣分高（48．0％左右）,配合力好,结铃性强,通风透光,皮棉产量比受体材料苏棉16号增产11．1％,品质两者相当。与其配置的杂交棉组合衣分高,丰产性好,品质高于对照南农8号,有着很强的杂种优势。转＆和GNA双价基因抗虫棉种质系BGsm16的成功选育,加强转基因棉花的抗虫能力,拓宽了抗虫谱,并有助于延缓害虫对抗虫棉产生抗（耐）性;同时改变了传统对常规抗虫棉衣分低的认识,丰富了抗虫棉种质资源,对选育高衣分杂交抗虫棉有良好的应用前景。     

转晚期胚胎发生丰富蛋白(LEA)基因棉花及抗旱性分析

本研究将紫杆柽柳(Tamarix and rossowii Litv)晚期胚胎发生丰富蛋白(LEADQ663481)基因导入新疆早熟棉新陆早18号,通过冬季海南加代、夏季新疆继代的方法获得T3代转基因棉花株系。从40株大田卡那霉素检测阳性植株中,经过PCR筛选证明3株为阳性的转化株。在室内条件下,对转化株幼苗进行水培实验,并用12%(w/v)PEG-6000处理12h模拟干旱胁迫。结果显示,干旱胁迫之后,转LEA基因棉花基因表达量显著增加;丙二醛生成量显著降低;游离脯氨酸、可溶性糖、可溶性蛋白的生成量显著增加;棉花表型分析也证明了转LEA基因棉花抗旱性有提高。培养45d后,转LEA基因棉花的生物量高于非转基因棉花。本研究结果表明,在干旱胁迫条件下,转LEA基因棉花表现出了优良的生长和生理优势,显示出转LEA基因棉花能提高棉花的抗旱能力。