Construction of cDNA library of cotton mutant Xiangmian-18 library during gland forming stage.
Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. This ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. Here we described a simple and efficient method for constructing a normalized cDNA library from a cotton mutant, Xiangmian-18, during its pigments gland forming stage. It combined switching mechanism at 5'-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN) normalization methods. In a model experiment, double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer of the original library was 5.86x10(5)cfu/ml in this library. Electrophoresis gel results indicated the fragments ranged from 800bp to 2kb, with the average size of 1400bp. Random picking clones showed that the recombination rate was 94%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of pigments gland cottons.
Xie
Yong-Fang
YF
Key Laboratory for Biomechanics and Tissue Engineering under the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.
Wang
Bo-Chu
BC
Li
Biao
B
Cai
Ying-Fan
YF
Xie
Lei
L
Xia
Yu-Xian
YX
Chang
Ping-An
PA
Jiang
Huai-Zhong
HZ
D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D015723:Gene Library; D003368:Gossypium / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012639:Seeds / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger