Inheritance of resistance toHelicoverpa armigera of 3 kinds of transgenicBt strains available in upland cotton in China

There are 3 kinds of transgenicBt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenicBt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenicBt cotton strains toHelicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenicBt strains shows that they may be inserted in the same chromosome. F₁ hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenicBt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.     

Commercial production of transgenic Bt insect-resistant cotton varieties and the resistance management for bollworm (Helicoverpa armigera Hubner)

There are currently three kinds of transgenic Bt insect-resistant cotton germplasm lines, Shanxi 94-24, Zhongxin 94 and R19, in China. They showed high resistance to the neonate larvae of bollworm (Helicoverpa armigera). Transgenic Bt insect-resistant cotton varieties or hybrids have been bred using the three kinds of germplasm lines as parents. Our researches reveal that there exist different expressions in resistant level at different developmental stages in the three categories of germplasm lines. When neonate larvae are fed with leaves of cotton plant at the seeding stage with less than 10 leaves on the main stem, the mortality of the neonate larvae is 100%, but the resistance level will decline at later season. When Bt gene has been transferred to the cotton genome, it can be steadily transferred to the progeny, the level of resistance to bollworm keeps fundamentally uniform. Such insects as tobacco budworm (Heliothis virencens) in laboratory directive selection are very apt to produce resistance to the Bt insecticidal crystal protein. From the present crop system of cotton region in the Yangtze and Yellow River Valleys, and the expression characteristic of transgenic Bt resistant cotton, we suggest that the resistance to toxin protein in bollworm is not apt to be produced if the transgenic Bt insect-resistant cotton varieties are released and grown in the regions except in the Xinjiang cotton region. The managing strategies to delay or retard the resistance are discussed.     

Inheritance of the delayed gland morphogenesis trait in Australian wild species ofGossypium

Five Australian wild cotton species with the delayed gland morphogenesis trait, as well as G. arboreum, G. davidsonii and four different gland genotypes of G. hirsutum, Gl₂Gl₂Gl₃Gl₃, Gl₂Gl₂gl₃gl₃, gl₂gl₂Gl₃Gl₃, and gl₂gl₂gl₃gl₃, were used in this experiment and 10 interspecific hybrids were obtained by the crossing among them. According to the gland expression on the seeds and plants of the interspecific hybrids, the inheritance of the delayed gland morphogenesis trait of Australian wild cotton species was opened out as follows: (ⅰ) the inheritance of the delayed gland morphogenesis trait was almost the same among the 5 Australian wild cotton species, and the gene or genes which controlled this trait may be located in the same loci. (ⅱ) The glandless seed trait of the Australian wild cotton species was dominant over the glanded seed trait of G. arboreum, a genome A species, and the seeds of interspecific hybrid F₁ between them were glandless. However, it was recessive over the glanded character of genome D species, G.davidsonii, and their F₁ was a typical glanded one. (ⅲ) The glandless seed trait of the Australian wild cotton species was recessive or incomplete dominant over the glanded cotton but dominant over the glandless cotton of G. hirsutum, and the glandless genes (gl₂gl₂gl₃gl₃) of upland cotton had great weakening effect on the glanded plant trait of the Australian wild cotton species on the other hand. For the two main glanded genes of upland cotton, the delayed gland morphogenesis trait of the Australian wild cotton species was dominant epistatic over glandless genes, gl₂gl₂gl₃gl₃, and one of the glanded genes, Gl₂Gl₂, but was recessive epistatic over the other glanded gene, Gl₃Gl₃. Therefore, it is much convenient to use Gl₂Gl₂gl₃gl₃ as the upland cotton parent in the interspecific hybridization and backcrossing afterward, in order to produce the upland cotton germplasm with glandless seeds and glanded plant trait.     

Variation induced by DNA rearrangement in a transgenicBt+CpTI cotton strain

In the development of transgenicBt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T₄ strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2–1/3 as large as that of the wild transgenicBt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n = 52; however, there are 4–8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCR amplification revealed thatBt andCpTI genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant phenotype might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred. Further analysis is ongoing.     

Insecticidal activity of residual Bt protein at the second trophic level

Since the first commercial release of transgenic crop expressing genes from Bacillus thuringiensis (Bt), there have been concerns about its potential impact on the environment. Research has focused on the ecological effects from second exposure to Bt prot…     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying Cry1Ac, Cry1Ie or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of Cry1Ac and Cry1Ie proteins were 0.173% and 0.131% of the total proteins, respectively. Cry1Ac protein content was 0.182% and Cry1Ie protein con- tent was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm (Helicoverpa armigera). The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and Cry1Ac-resistant cotton bollworm than those carrying Cry1Ac or Cry1Ie alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resis- tance to transgenic crops.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.     

种植转Bt水稻对固氦细菌多样性和固氮酶铁蛋白基因nifH的水平转移的影响

通过对具有不同种植年限和不同种植强度的转Bt基因水稻与非转Bt基因水稻土壤中的细菌以及固氮细菌群落结构进行研究,发现转Bt水稻的种植可能会影响土壤中微生物群落的多样性,但是这种影响的可能只是暂时的,通过对测量种植水稻的芽长实验也得出相似的结论．另外,根据16SrDNA基因构建的系统发育进化树揭示了本实验分离的固氮细菌的遗传多样性,发现实验土壤中的固氮细菌主要分为放线菌门（Actinobacteria）（92％）和α-变形菌门（α-Pro—teobacteria）两大类．分离出的8株典型的固氮菌株,其16SrDNA基因和固氮基因nifH的序列两者的分布不一致,nifH的分布更为紧凑,为固氮基因可能发生了原位水平基因转移提供了证据．     

Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development ofHelicoverpa armigera

The resistance ratio ofHelicoverpa armigera to Cry1 Ac insecticidal protein fromBacillus thuringiensis (Bt) is 13.1- and 3.02-fold after 18 generations of selection by transgenic tobacco expressing Bt or two (Bt and CpTI) insecticidal protein genes, in which the average corrected mortality for each selection treatments is about 60%. The mortality of selected population by transgenic Bt gene tobacco is significantly lower than the control strain when fed on transgenic tobacco plants. The mortaltty of the selected population by transgenic two genes tobacco was not significantly different from the control strain. This is the first experiment under laboratory condition which has proved that transgenic two genes tobacco could significantly delay resistance development ofH. armigera compared with one gene.     

The Recent Advances in Plant Protection Researches of China

There are eight examples briefly given in this paper, namely, (1) Polymyxa graminis and the cereal viruses it transmits; (2) the geographical types and facultative migration of cotton bollworm as well as the safety of Bt transgenic cotton; (3) development of crop near-isogenic lines with resistance to diseases; (4) molecular-biological researches induced resistance of rice by infection of blast fungus;(5) to use cytological and molecular-biological techniques for breeding wheat varieties resistant to barley yellow dwarf virus; (6) mass rearing and field releasing of Microplitis mediator for cotton bollworm control; (7) identification and recombination of insecticidal crystal genes of Bacillus thuringiensis; and (8) interplanting of diverse resistance rice varieties for sustainable control of blast disease; which reflect the general situation of recent advances in plant protection researches of China.     

Identification of RAPD marker linked with fertility-restoring gene of cytoplasmic male sterile lines in upland cotton

Bulked segregant analysis was employed to construct two mixed DNA pools to screen the RAPd marker linked with the fertility-restoring gene(Rf _i) of upland cotton. A total of 425 arbitrary 10-mer oligonucleotide primers were screened on two DNA pools, bulked male fertile and sterile DNAs isolated from BC₃ segregating population of (0-613-2R X Simian No. 3). Three primers produced repeatable polymorphisms between the paired bulks and their parents. DNA was extracted and amplified with these three primers for 92 plants of (Zhong 12A-1 × 0-613-2R)F₂. Based on the male fertility scoring and RAPD amplification, it is found that one RAPD marker fragment designated OPV-15₃₀₀ was linked with the fertility-restoring gene (Rf₁) with a recombination value of 13.0±2.57%.     

Breeding,introgression and inheritance of delayed gland morphogenesis trait from<Emphasis Type="Italic">Gosspium bickii</Emphasis> into upland cotton germplasm

Cotton is a valuable economic crop. The cottonseedafter ginning is made up of fuzz, kernel and hull, amongwhich kernel constitutes about 50% to the seed weight.Cottonseed kernel of G. hirsutum contains about 40% ofprotein and more than 35% of cottonseed oil, which obvi-ously is a potential rich source of high quality protein andedible oil supplement[1,2]. However, the utilization of cot-tonseed is limited by the presence of gossypol and its de-rivatives in seeds of ordinary glanded cotton cult…     

对棉花三系在配制杂种中应用价值的研究

将五个陆地棉恢复系和三个雄性不育系进行杂交，得到１５个杂种Ｆ１；并对杂种Ｆ１的重要农艺性状及纤维品质进行了研究．对试验数据的方差分析表明：有关铃重、衣分、纤维长度和整齐度方面杂种Ｆ１间的差异达到极显著的水平，而在皮棉产量、单株吐絮铃数上的差异却是不显著的．不育系中Ａ和ＰＡ在铃重、衣分和绒长上的一般配合力是高度显著的．恢复系４Ｒ—１６在铃重、衣分和纤维整齐度的一般配合力也都达到了极显著的水平．这些试验结果对以后的研究和选育强优杂种是有用的。     

转Bt+CpTI基因抗虫棉品种SGK321性状分析和种质利用

SGK321是以"石远321"为受体,导入Bt CpTI双价抗虫基因后育成的抗虫棉品种。SGK321霜前皮棉产量水平高于常规对照品种,接近杂交对照品种。其早熟性较好,生育期短于对照品种,霜前花率高于对照品种,第一果枝节位适中。抗虫性明显优于一般Bt抗虫棉,三代棉铃虫3龄以上幼虫存活率和幼虫株率与对照相比明显下降;用SGK321棉叶饲养棉铃虫,5龄以下幼虫不能化蛹和羽化;对抗性棉铃虫具有较强的抗虫性,尤其是对大龄幼虫的正常生育阻碍作用明显,对幼虫致死快,致死率高;还可延缓棉铃虫抗性的产生。稳产适应性较好、抗逆性强。具有丰富的遗传基础,一般配合力和特殊配合力较高,2001-2006年6年间作为种质资源被中国众多育种机构利用,已培育出72个双价抗虫棉品种(系),其中24个品种通过国家及省级审定,18个品系参加了各级试验;育成抗虫、抗病、大铃、高衣分、优质等不同类型种质材料237份。对中国棉花生产与品种改良产生了重要影响。     

Integration and inheritance stability of foreignBt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R₃, R₄ and R₅ generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of ₃, R₄ and R₅ generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.     

牛奶中苏云金芽胞杆菌的分离与鉴定

采用醋酸钠．抗生素分离法对超市牛奶样品进行苏云金芽胞杆菌（Bacillus thurgngiensis,Bt）的分离,获得2株Bt菌株,分别命名为BtBRC—LJ1和BtBRC-LJ2。利用PCR方法对Bt BRC-LJ1和BtB RC-LJ2菌株幼皿和nheC肠毒素基因的分布进行检测。结果显示,这2个菌株均含有这两种肠毒素基因。     

Construction of a linkage map and QTL mapping for fiber quality traits in upland cotton (Gossypium hirsutum L.)

With the development in spinning technology, the improvement of cotton fiber quality is becoming more and more important. The main objective of this research was to construct a high-density genetic linkage map to facilitate marker assisted selection for fiber quality traits in upland cotton (Gossypium hirsutum L.). A genetic linkage map comprising 421 loci and covering 3814.3 cM, accounting for approximately 73.35% of the cotton genome, was constructed using an F2 population derived from cross GX1135 (P 1 )×GX100-2 (P 2 ). Forty-four of 49 linkage groups were assigned to the 26 chromosomes. Fiber quality traits were investigated in F2 population sampled from individuals, and in F2:3 , and F2:4 generations sampled by lines from two sites and one respectively, and each followed a randomized complete block design with two replications. Thirty-nine quantitative trait loci were detected for five fiber quality traits with data from single environments (separate analysis each): 12 for fiber length, five for fiber uniformity, nine for fiber strength, seven for fiber elongation, and six for fiber micronaire, whereas 15 QTLs were found in combined analysis (data from means of different environments in F2:3 generation). Among these QTLs, qFL-chr5-2 and qFL-chr14-2 for fiber length were detected simultaneously in three generations (four environments) and verified further by combined analysis, and these QTLs should be useful for marker assisted selection to improve fiber quality in upland cotton.     

新疆棉花枯萎病菌酯酶同工酶的研究

利用聚丙烯酰胺凝胶电泳测定新疆棉花枯萎病菌的酯酶同工酶,结果表明,供试的３０个代表性菌株的酯酶同工酶有４种类型的主酶带,类型１有Ｅ２１,Ｅ２０,Ｅ１９,Ｅ６,Ｅ５ ５条主酶带,包括２５个供试菌株;类型有Ｅ２１,Ｅ２０,Ｅ５４个主酶带,包括１个供试菌析对照３析标准菌株的酯酶同工酶及１９９７年新疆棉花枯萎病菌生理小种鉴定结果,可以看出：新疆棉花枯萎病菌生理小种与酯酶同工酶有显著性相关性。用生化的方法再     

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2—3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6—7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6—7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3︰1 segregation law. 8 homozygous R₁ transgenic plants harboring 2—3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.     

转基因植物的非靶标效应——以转Bt基因棉为例

本文综述了转Bt基因棉的非靶标效应的研究进展,在此基础上分析了转基因植物非靶标效应研究的研究特点和不足,并对这类研究的发展方向进行了展望.