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Cloning of Fiber-specific Gene and Its Promoter from Cotton

Author: AnJun
Tutor: WangZhiXing
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: mRNA differential display tissue specific promoter
CLC: S562
Type: Master's thesis
Year: 2008
Downloads: 135
Quote: 1
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Abstract

China is the largest cotton producing and consuming country in the world, but the fiber quality is not as good as that of the U.S. and other cotton-producing countries. So it’s an urgent task to develop new cotton varieties with improved fiber quality. Through traditional breeding techniques, it is very difficult to improve the fiber quality and increase cotton production simultaneously.Two strategies can be used to improve fiber quality through genetic engineering. One strategy is isolating and identifying fiber quality-related genes, study the molecular mechanisms of cotton fiber development, then increase and decrease the expression of these quality-related genes. Another way is selecting some useful genes from other plants and transforming them into cotton. CaMV 35S promoter can make foreign gene expressed stably in cotton, but the gene is expressed in the whole plant result in a large consumption of material and a bad affection of plant growth. Therefore, the cloning of fiber-specific promoter will be an effective tool for improving the quality of cotton fiber through genetic engineering and has a important and practical significance.In this study, upland cotton Su-12 was use as a material and specific expression segments of cDNA in cotton fibers were separated from those in leaves by mRNA differential display. 10 cDNA segments which are specific expression in cotton fibers were identified, full -length cDNA of GhTIP and part of its promoter were cloned , the results are as follows:（1） Cotton mRNA differential display system optimization:Total RNA was extracted from leaves and fibers using CTAB method. Concentration of primers, dNTP and Mg²⁺, the three key factors of mRNA differential display technology, were adjusted and the differential display system was optimized for cotton fibers and leaves.（2） 24 different cDNA segments were screened out through mRNA differential display technology. 10 fibers specific cDNA segments were identified by screening reverse dot hybridization and preliminary analysis was conducted through NCBI Blastx and Blastn.（3） On the basis of fiber specific cDNA segments, a full-length cDNA was cloned. Using the tool of Blastn, it is found that the cDNA is the gene of Gossypium hirsutum aquaporin gamma-TIP and the DNA sequence contained two introns.（4） Part of the GhTIP 5′upstream sequence was cloned using chromosome walking technology. Plant CARE analysis showed that the sequence contains some promoter components, such as CAAT - box, TATA– box, HSE, Sp1 and GT1（5） Through the ligation of GhTIP 5′upstream sequence and gus gene, plant expression vector was constructed. Gus assays of transgenic tobacco exhibited the promoting function of this fragment.

Cloning of Fiber-specific Gene and Its Promoter from Cotton

Abstract

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