Cloning Of PhaB And PhaC Genes And Construction Of Their Cotton Fiber Expression Vectors

Cotton, as the premier natural fiber for textile application, is one of the most important cash crops in our country. It ranks China the first both in planting acreage and in total yield in the world. The amount of cotton export in our country also makes the first and cotton also takes a considerable part in earning foreign profits. Like other crops, the development of cotton industry is definitely depend on the development and release of elite cultivars. In the last several decades, cotton breeders contributed a lot to cotton breeding and many elite cultivars were released.Although great achievements have been made in cotton breeding, there are still some deficiencies. In the past, great attention was paid to the yield and fiber length, while the improvement of fiber inner quality was ignored. Thus, all the cultivars planting in our country are high yield and long fiber, but fiber quality is poor. But with the development of textile technique and the innovation of textile machines, and what's more, with the improvement of the people's living standards, high quality fiber cotton cultivars are in great need. Consequently, improving fiber quality has become the main purpose in present cotton breeding.Poly-3-hydroxybutyrate (PHB) is produed by many genera of bacteria. It is an archetype that is a natural biodegradable themplastic.PHB is light,well flexibility,plasticity and resistance to ultraviolet radiation. If PHB is produced in cotton fiber lumen,the thermal characteristics and shrink properties of cotton fiber will be greatly improvedThere are 3 main task tasks in the present research. The first is to clone the two aimed genes (phaB and phaC) and two cotton fiber-specific promoter(FbL2A and LTP12).then analysis their sequences.The construction of plant expression vector which cotains phaB and phaC is the second. The third is the congstruction of their bacteriam expression vectors and how to induce them to express in the Escheri chia colis.Two PHB synthase genes and two fiber-speacific promoters was cloned by PCR Amplification.According the results of sequence analysis: there are two TATA boxes in FbL2A,they are 55bp and 236bp before the initiator coadon separately.There is no difference between the cloned sequence and the sequence from GenBank .PromoterLTP12 is 771bp in length and there is a TATA box at the site of 640bp,the consensus between the cloned sequence and sequence from reference is 98.2%.The gene phaB has 751 base pairs and encodes 247 amino acids.the percentage ofconsensus between cloned sequence and sequence from reference is 99%.phaC is 1770bp in length and encodes 594 amino acides . There is a 99% consensus between the clonewd phaC sequenced and sequenced from reference.pBFPBPC and pBLTPPBPC are plant expression vectors which both cotain PHB. Both vectors contain the two genes ,phaB and phaC , while in the former under the control of promoter FbL2A,and in the latter under the control of LTP12.pETPB and pETPC are all bacteriam expression vectors,The former contain phaB ,and the latter contains phaC.the two vectos are all transferred into E.coli. Protein which is approximately 32KD appeara while pETPB was induced by IPTG.The result is in accord with actual fact.No protein was dected while phaC was induced.