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Chinese Science Bulletin 2000年第14期45卷 1249-1257页

作者： ZHANG Tianzhen & TANG CanmingKey Laboratory of Crop Germplasm & Breeding, Ministry of Agriculture Department of Agronomy, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Crop Germplasm & Breeding Ministry of Agriculture Department of Agronomy Nanjing Agricultural University Nanjing China

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

关键词： transgenic Bt insect-resistant cotton Helicoverpa armigera resistance spatial and temporal expression insect resistance management.

Variation induced by DNA rearrangement in a transgenic Bt+CpTI cotton strain

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Chinese Science Bulletin 2001年第7期46卷 559-562页

作者： Wangzhen Guo Tianzhen Zhang Xiefei Zhu Xiaoling Yuan Key Laboratory of Crop Germplasm and Breeding Ministry of Agriculture Department of Agronomy Nanjing Agriculture University Nanjing China

In the development, of transgenic Bt + 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 transgenic Bt + 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. PCRamplification revealed that Bt and CpTl 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 pheno-type 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.

关键词： transgenic insect-resistant cotton mutant variation DNA rearrangement.

Transgenic Tobacco Plants With Efficient insect Resistance

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Science China Chemistry 1994年第12期37卷 1479-1488页

作者：李太元田颖川秦晓峰莽克强李文谷何永刚沈蕾 Institute of Microbiology Academia SinicaBeijing 100080PRC Institute of Entomology Academia SinicaShanghai 200025PRC

insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(*** gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of *** *** stripes of tobacco plant ***89 widelygrown in China were transformed with *** LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were *** test with tobacco *** showed that insect-resistant *** could be obtained from the regenerated plantstransformed with *** of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin ***,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that *** gene and the character of insect resistance have been inherited in the ***-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.

关键词： B. t. toxin gene transgenic tobacco plants insect-resistant homozygote.

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

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Science China(Life Sciences) 1996年第3期39卷 320-328页

作者：周兆斓朱祯刘春明张海涛肖桂芳李向辉 Institute of Genetics Chinese Academy of Sciences Beijing 100101 China

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

关键词： rice cDNA library oryzacystatin tfaiol proteinase inhibitor insect-resistant gene sequence analysis.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in ***

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High Technology Letters 1996年第2期2卷 89-94页

作者：周兆斓 Institute of Genetics Academia SinicaBeijing 100101P.R.China

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in *** the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins *** papain-inhibitory activity of the expressed OC was further confirmed.

关键词： Rice cDNA library Thiol proteinase inhibitor insect-resistant gene Sequence analysis High level expression