Cloning, Expression And Characterization Of Three Novel Genes Relation To β-oxidation Of Fatty Acids In Upland Cotton

As a major source of fibers, cotton is an important economic crop and plays an important role in the global economy. With the development of textile technology and the rasied requirement of society, it is urgent to breed and cultivate cotton varieties with high yield production, super fiber quality and high stress tolerance. It’s become a hot spot of research as the metabolism of fatty acidβ-oxidation in plant playing a significant role in regulating development and growth and responding to the the stress of environment. Numerous experiments of mutant proved that fatty acidβ-oxidation could provide seeds carbon and energy before photosynthesis happened, also effected the signal transduction systerm and development of plant. Consequently, researches of fatty acidβ-oxidation are beneficial to find out the molecular mechanism of plant adaptability and stress tolerance, furthermore, it provides foundation to breed plant with stress-tolerant.The cole step of metabolism of fatty acidβ-oxidation in plant peroxidaseis that thio-alcoho end of acyl-CoA two carbon unit (acetylacyl-CoA) thiolasis again and again. The mechanism are catalyzed by the three core enzymes of theβ-oxidation cycle, namely acyl-CoA oxidase (ACX), the multifunctional protein (MFP) of which there are two isoforms,which exhibit hydratase, dehydrogenase, epimerase, andisomerase activities, and 3-ketoacyl-CoA thiolase (KAT). Our research focused on identifying enoyl-CoA hydratase (ECH), a enzyme with singal function catayze hydration; 3-ketoacyl-CoA thiolase (KAT) and acetoacetyl-CoA thiolases (ACAT) which catayze retrorse reaction. In this research these genes are cloned, identified and expression characterizied in upland cotton, ulteriorly, discover the relationship with stress-tolerance and germing, discuss their functions in development and abiotic stress response in cotton.The mainly results are as follows:(1) Contigging the homologous ESTs in upland cotton, we acquired GhKAT, GhACAT and GhECH genes. The full length of GhKAT was 1630bp, and its open reading frame (ORF) was 1334bp, encoded a polypeptide containing 455 amino acids. The protein molecular weight (Mw) was 47.975 kDa and a calculated isoelectric point (pI) was 8.91. It had a typical thiolase domain between 55-436 in the amino acid position. The full length of GhACAT was 1500bp, and its ORF was 1221bp, encoded a polypeptide containing 407 amino acids. The protein molecular weight (Mw) was 41.440 kDa and a calculated isoelectric point (pI) was 6.02. It had a typical thiolase domain between 28-407 in the amino acid position. The full length of GhECH was 919bp. its ORF was 705bp, encoded a polypeptide containing 235 amino acids. The protein molecular weight (Mw) was 25.119 kDa and a a calculated isoelectric point (pI) was 8.10 It had a typical crotonase domain between 28-407 in the amino acid position.(2) Quantitative RT-PCR found that this three genes expressed in all of the vegetative organs, and they were houskeeping genes. But they were highest expressed in 25 DPA ovules. Furthermore, these genes had lower expression in roots, stems, cotyledons, leaves, fibers.(3) The analysis of expression profile induced by the abiotic stress indicated that GhKAT could instantaneously induced by ABA, and its transcripts were accumulated with treated, but declined after 12 hours. Moreover, its expression was not increased when treated with drought, cold and wounding. The expression of GhACAT and GhECH were declined when treated with ABA, but they could be induced by drought, wounding, expect cold. Furthermore, the accumulation responsed to abiotic stress of GhECH was earlier and quicker contrasted to GhACAT.(4) We predicted their N terminus signal peptide via software signalP3.0 process. They all had no typical signal peptide, proved that they were non-secreting proteins. Subcelluar localization research of GhECH proved that it was a protein acted on nucleolus and membrane, possibly expressed on membranes.(5) Using the BC1 mapping population derived from [(TM-1×Hai 7124)×TM-1], GhACAT was localized on chromosome 11 (A11), between the SSR marker NAU3485 and E13M13; while GhECH was localized on chromosome 14 (D2), between the SSR marker Gh329 and BNL1681.