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Cloning and Characterization of Three Genes Related with Auxin in Gossypium Hirsutum L.

Author: ZuoYiBin
Tutor: GuoWangZhen
School: Nanjing Agricultural College
Course: Crop Genetics and Breeding
Keywords: cotton adenosylhomocysteine hydrolase IAA/auxin-responsive protein ethylene responsive element binding protein
CLC: S562
Type: Master's thesis
Year: 2006
Downloads: 111
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Abstract

Cotton is the most prevalent natural fiber used as the raw materials in textileproduction, and is one of the most important oil crops in the world. Cotton fiber is knownone of the natural resources containing the highest cellulose purity. Auxin plays animportant part in the entire growth process of cotton. So cloning the genes related to auxinin Gossypium hirsutum L may play an important role not only in studying plant celldevelopment, but also providing the information to biological study of cotton fiberdevelopment.Three cDNA clones were separated from developmentally different cotton fiber poolof premium material 7235 library. One of the clones encodes adenosylhomocysteinehydrolase（designated as GhSAHH）. The antisense expression of said gene could increasethe content of free CTK in cells, inducing the defence of antihardiness of plant. The insertfragment of the cDNA clone is 1598bp, and a 318-bp-long-upstream fragment was obtainedvia 5’RACE technique. After integrated with the original sequence, we acquire a cDNAsequence accounting for 1916bp. Its open reading frame is 1458bp, and encodes apolypeptide containing 485 amino acids, and the Mw of the deduced amino acid is 53.2Da,and pI is predicted to be 5.69. There exists an in-frame stop codon in the upstream. There isno signal peptide among this deduced amino acid, so it maybe an intracellular enzyme.BLAST analysis indicates that it shares a high homological identity with otheradenosylhomocysteine hydrolase from other plants. The protein of GhSAHH, GhSAHH hada 83％identity to the Medicago sativa adenosylhomocysteine hydrolase （GenBankAccession number:ALFMSA2S）. Judging from its expression characters, GhSAHH isconstitutive expression in cotton cells, its expression quantity is comparatively high in theearly stage of fiber developmental period and reduces as fiber develops; it is also expressedin root, stem and leaf. Southern blotting analysis shows that there are one copies ofGhSAHH possibly in the genome of upland cotton; sub-genome A and sub-genome Dcontains each. We have used the BC₁ mapping population derived from the hybridizationbetween the upland cultivar TM-1 and the island cultivar Hai7124, further TM-1 asrecurrent as parent. GhSAHH was localized on the chromosome 20 respectively. Underwater stress, down regulated expression of GhSAHH could be observed in cotton leaf,inducing the defence of antihardiness of plant.The second clone, encodes auxin responsive protein （designated as GhARP）. The geneencodes an early responsive protein of the transduction of IAA signal. Its insert fragment is1696bp; its ORF is 1074bp and encodes a polypeptide including 357 amino acid residues. The predicted Mw of the deduced amino acid is about 38.9kDa, and its pI is 6.62. There isno signal peptide among this deduced amino acid, so it maybe an intracellular enzyme.BLAST analysis shows GhARP had a 85％identity to grape （Vitis vinifera） auxinresponsive protein （GenBank Accession number: AY082522）. GhARP is constitutiveexpression in cotton cells, and its expression quantity is comparatively high in root, stem,leaf and all stages of fiber, without distinct difference. Southern blotting analysis shows thatthere are many copies of GhARP possibly in the genome of upland cotton. GhARP’s senseantisense fragments were integrated intoplant expression vector（pBI121） with CaMV35S.The transgenic research on cotton is undertaking. Under water stress, the peak of GhARPexpression in cotton leaf was achieved after 6 days of stress, and then decreased gradually.The third clone, encodes ethylene responsive element binding protien（designated asGhEREBP）. Said gene encodes an important regulation protein in the process of ethylenesignal transduction. Its insert fragment is 1563bp; its ORF is ll91bp and encodes apolypeptide including 396 amino acid residues. The predicted Mw of the deduced aminoacid is about 38.9kDa, and its pI is 6.62. There is no signal peptide among this deducedamino acid, so it maybe an intracellular enzyme. BLAST analysis shows GhEREBP had a96％identity to Gossypium hirsutum ethylene responsive element binding protien（GenBank Accession number: AY817134）. GhEREBP is expressed predominantly infiber cells, and its expression quantity is comparatively low in the early stage of fiberdevelopmental period and increases as fiber develops; it is also expressed in root, stem andleaf. Southern blotting analysis shows that there are many copies of GhEREBP possibly inthe genome of upland cotton. GhEREBP’s sense antisense fragments were integrated intoplant expression vector（pBI121） with CaMV35S. The transgenic research on cotton isundertaking. Under water stress, the expression of GhEREBP in cotton leaf varies little.

Cloning and Characterization of Three Genes Related with Auxin in Gossypium Hirsutum L.

Abstract

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