| Human serum albumin (HSA) is the most abundant protein in huaman blood plasma, accouting for 60% of the total serum proteins. It also exists in tissue, body liquid, skin and lymph chamber, and forms the extravascular pool. Mature HSA is a single-strand non-glucoprotein, composed of 585 amino acids. HSA takes an important role in human bodies. For example, it keeps the osmotic pressure of blood and transports nutrients. So it's widely used in clinical practices, mainly used to treat exsanguine patients caused by shock, burn, surgery, and so on. In addition it's efficient to those suffered from chronical nephritis, hepatic diseases and malignancy. In this research, total RNA was extracted from fetal liver by the modified single-step method of acid guanidinium thiocyanate-phenol-chloroform (AGPC). Based on reported sequence of HSA cDNA, specific primers were designed to amplify its encoding region by RT-PCR. At the same time, Kozak sequence was added to the translation site, to strengthen initiation of translation. The fragent of cloned HSA cDNA is 1758bp. NCBI BLASTn analysis indicated that similarity of HSA cDNA sequence and the postulated ammonic acids sequence is 99.6% and 99.5%, compared with the reported sequence in GenBank. So the modified HSA cDNA with Kozak sequence was obtained.It's well known that rubber tree is rich of laticifers closely related with latex production. It's advantageous to be a plant bioreactor. Once the heterogeneous gene expresses in laticifers, the protein product can be easily harvest, isolated and purified. Thus it's necessary to recombine the heterogeneous gene with the promoter of laticifer-expression gene, and then the heterogeneous gene will express in laticifers. So the primary thing is to clone this kind of promoters. Rubber elongation factor (REF) is a rubber particle membrane-bound protein, Mr. 14.6kDa. Previous study indicates that REF is highly expressed in latex, accounting for 10~60% of the total protein in latex. REF is very important for polymerization of rubber molecules. Genomic DNA was extracted from light foliage by SDS method. Two fragments (1~378, 378bp; 73~378, 306bp) of REF promoter were amplified by PCR using different specific primers, and thensequenced. Compared with the reported sequence in GenBank, similarity of DNA sequence was more than 98%, so right sequences were cloned and modified. And many important cis-acting regulatory elements were found in the cloned sequence region by PlantCARE software analysis. REF promoter was predicted to be most probably induced by light, hot, gibberellin, ethenen or wound. To identify its laticifer-expression character, two transient-expression vectors (pIRl, pBIR2) were constructed. In the transient-expression vector, REF promoter sequence replaced CaMV35S promoter of pBI121. pBIRl and pBIR2 were separately transformed into light foliage of H. brasiliensis by bombardment. GUS histochemistry analysis indicated that pBIRl and pBIR2 had nearly the same results: GUS gene had been successfully transformed into the bombarded foliage. And the blue dots were found along with the veins. But there wasn't any blue dot at other parts of the positive explants. At the same time, no blue dots were found in the foliage bombarded by the microcarriers coated with ddH2O. This result showed that GUS was transiently expressed in the foliage, and they were of laticifer-expression. The efficiency of transformation, caused by different bombardement distances (3.0cm, 6.0cm, 9.0cm), was also valued. Bombardment with 9.0cm is most efficient, 80% positive explants obtained from pBIRl, and 77.5% from pBIRl. Each sample was bombarded 2 times. When the distance increases from 3cm to 9cm, the percent of positive explants climbs. Therefore, the distance of 9.0cm and bombardment for two times each treat are appropriate parameters to transform heterogeneous gene into foliage explant of H. brasiliensis.A perfect expression vector is essential for the heterogeneous gene to efficiently express in laticifers of rub...
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