Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts.
An optimized vip3A gene, designated as vip3A* was chemically synthesized and a thi1 gene chloroplast transit peptide coding sequence was attached to its 5' end to produce the tvip3A*. vip3A* and tvip3A* genes were transformed into Gossypium hirsutum cv. Zhongmiansuo35. Of 42 independent transformants, 36 were positive for the vip3A* or tvip3A* gene. Four independent transgenic T1 lines with single-copy insertions and unchanged phenotypes (CTV1 and CTV2 for tvip3A*, and CV1 and CV2 for vip3A*) were selected by Southern blotting, and subjected to an insect bioassay and field assessment. Four homozygous T2 transgenic lines were then selected and the amount of expressed Vip3A* protein was determined by western blotting and ELISA. The protein concentrations of CTV1 and CTV2 were about three-fold higher than those of CV1 and CV2. As expected, the Vip3A* protein of CTV1 and CTV2 were transported to the chloroplasts, where they accumulated. The Vip3A* protein concentration in the chloroplasts of CTV1 and CTV2 was about 15-fold of that of CV1 and CV2. All four transgenic lines showed 100% mortality against fall armyworm (Spodoptera frugiperda) and beet armyworm (Spodoptera exigua) by insect bioassay. Moreover, CTV1 and CTV2 exhibited 100% mortality against cotton bollworm (CBW, Helicoverpa zea), whereas CV1 and CV2 showed 75.0% and 72.5% mortality against CBW, respectively. The field bioassay indicated that CTV1 and CTV2 were more resistant to CBW than CV1 and CV2. Our results suggest that the two tvip3A* transgenic lines (CTV1 and CTV2) can be used to develop insect-resistant cultivars and could be used as a resource for raising multi-toxins-expressing transgenic cotton.
Jiahe
J
Wu
Xiaoli
X
Luo
Xiangrong
X
Zhang
Yuejing
Y
Shi
Yingchuan
Y
Tian
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D003368:Gossypium / Q000235:genetics* / Q000469:parasitology*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics* / Q000469:parasitology*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000469:parasitology*; D018411:Spodoptera
D001426:Bacterial Proteins; C447139:Vip3A protein, Bacillus thuringiensis
Protein Expr Purif. 2002 Oct;26(1):82-8
12356474
Theor Appl Genet. 1992 Mar;83(5):645-9
24202683
J Econ Entomol. 2008 Aug;101(4):1244-52
18767734
J Invertebr Pathol. 2007 Jul;95(3):227-30
17475274
Annu Rev Entomol. 2002;47:845-81
11729093
J Econ Entomol. 2007 Feb;100(1):180-6
17370826
Appl Environ Microbiol. 2001 Dec;67(12):5855-8
11722946
Sheng Wu Gong Cheng Xue Bao. 2002 Nov;18(6):687-92
12674638
Anal Bioanal Chem. 2008 Jan;390(1):377-87
17994293
Nat Biotechnol. 2001 Jan;19(1):71-4
11135556
Biochem Biophys Res Commun. 2006 Jan 27;339(4):1043-7
16337146
Biochem Biophys Res Commun. 1990 Jul 31;170(2):665-72
2116794
Plant Mol Biol. 1987 Mar;10(2):105-16
24277496
J Econ Entomol. 2002 Feb;95(1):14-21
11942749
FEMS Microbiol Lett. 2005 Mar 15;244(2):353-8
15766790
Theor Appl Genet. 2002 Aug;105(2-3):258-264
12582527
Anal Biochem. 1976 May 7;72:248-54
942051
J Cell Sci. 2003 Jan 15;116(Pt 2):285-91
12482914
Plant Mol Biol. 2001 Aug;46(6):639-50
11575719
Annu Rev Entomol. 2002;47:501-33
11729083
Appl Environ Microbiol. 2008 Jan;74(2):462-9
18024681
J Econ Entomol. 2003 Aug;96(4):1031-8
14503572
Plant Cell Rep. 1987 Jun;6(3):231-4
24248660
Appl Environ Microbiol. 2009 Apr;75(7):2236-7
19181834
Appl Environ Microbiol. 1997 Feb;63(2):532-6
9023933
J Invertebr Pathol. 1990 Sep;56(2):258-66
2273290
Proc Natl Acad Sci U S A. 1996 May 28;93(11):5389-94
8643585
Appl Environ Microbiol. 2003 Aug;69(8):4648-57
12902253