Cloning of Ds MAPK Gene and Construction of Antisense Expression Vector

In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs MAPK gene into downstream sequence of 35S promoter of plant expression vector, an antisense expression vector of Ds MAPK gene was successfully constructed and introduced into D. salina cells by LiAc/PEG-mediated method. The expression of Ds MAPK gene in transgenic D. salina was analyzed by semi-quantitative RT-PCR method. The results showed that the expression of Ds MAPK gene in D. salina was significantly inhibited at the transcriptional level. The study laid the foundation for further identification of the function of Ds MAPK gene.

Construction of Plant Antisense Expression Vector with Defective in Anther Dehiscence1 Gene Fragment of Chinese Kale

A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.

Increasing cellulose production and transgenic plant growth in forest tree species

Cellulose is one of many important polymers in plants. Cellulose is made of repeat units of the monomer glucose. Cellulose is a major industrial biopolymer in the forest products, textile, and chemical industries. It also forms a large portion of the biomass useful in the generation of energy. Moreover, cellulose-based biomass is a renewable energy source that can be used for the generation of ethanol as a fuel. Cellulose is synthesized by a variety of living organisms such as plants and algae. It is the major component of plant cell walls with secondary cell walls having a much higher content of cellulose. The relationship between cellulose and lignin biosynthesis is complicated, but it is confirmed that inhibition of lignin biosynthesis in transgenic trees will increase cellulose biosynthesis and plant growth. Cellulose accumulation may be increased by down-regulating 4-coumarate:coenzyme A ligase (4CL, EC 6.2.1.12) as shown in transgenic aspen. There is no similar reports on down-regulating 4CL in transgenic conifers. Based on our established Agrobacterium tumefaciens-mediated transformation system in loblolly pine, we are able to produce antisense 4-CL transgenic loblolly pine which is predicted to have increas- ing cellulose accumulation. The overall objective of this project is to genetically engineer forest tree species such as loblolly pine with re- duced amount of lignin and increased cellulose content. The research strategy includes: (1) isolate the 4-coumarate:coenzyme A ligase gene from loblolly pine seedlings by reverse transcription-polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR) techniques from the cDNA library; (2) construct binary expression vectors with antisense 4CL coding sequences and introduce antisense constructs of the 4-coumarate:coenzyme A ligase gene cloned from loblolly pine into the loblolly pine to down regulate the 4-coumarate:coenzyme A ligase gene expression; (3) study the effect of the antisense transgene expres- sion on lignin content, cellulose accumulation, and loblolly pine biomass; and (4) select fast growth and high cellulose accumulation transgenic loblolly pine lines for future commercial application.

EFFECTS OF ANTISENSE EPIDERMAL GROWTH FACTOR AND ITSRECEPTOR RETROVIRAL EXPRESSION VECTORS ON CELLGROWTH OF HUMAN PANCREATIC CARCINOMA CELL LINE

A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and / or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7 / AS-EGF, PC-7 / S-EGFR, PC-7 / AS-EGFR and PC-7 / pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7 / AS-EGF and PC-7 / AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7 / AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7 / AS-EGFR cell line.