Studies On Construction Of IhpRNAi Plant Expression Vector Of Potato GBSS Ⅰ Gene Driven By Patatin Promoter And Transformation Of Potato

Starch is the second largest carbohydrate only second to cellulose. There are about 17% starch in potato (Solanum tuberosum L.) tubers. Potatoes are the suitable crop to produce starch, which has been the important raw materials in both food and industrial applications. Starch consists of amylose and amylopectin, High- amylopectin starch shows the better stability ,solubility, viscosity and transparency , widely used in adhesive tape, adhesives in industrial and papermaking, textile, architecture, petroleum and so on. High-amylose starch has very low water-solubility, being easy to retrogradate and diffcult to gelatinate. its property is similar to fiber, and it is potential raw material to produce biodegradable plastic film.Therefore, it has great significance to cultivate high- amylopectin or high-amylose potato varieties.Research has shown that the key enzymes which are controlling starch biosynthesis are Adenosine 5′diphosphate glucose pyrophosphorylase, Starch synthase, Starch-branching enzyme and Starch debranching enzyme. The development of molecular biology techniques makes it possible to regulate the activity of key enzymes in starch biosyntheses and modify the content and structure of starch by genetic engineering , especially the RNA interference (RNAi) technique can significantly affect the expression of target gene, then chang the content of amylose and amylopectin and their radio.In accordance with the principle of RNAi expression, we chose the conserved sequences (772-1313bp) of Granule-bound starch synthaseⅠ(GBSSⅠ), which is controlling amylose synthesis to construct the ihpRNAi plant expression vector containing gbss A-intron-gbss B, then transfromed into potatoes by use of Agrobacterium tumefaciens in order to make GBSSⅠgene silence and get new germplasm resources of potatoes which have very little amylose or have non-amylose. we have done some works as follows:1. The core conserved sequence of GBSSⅠgene was amplified from potato tubers. The nucleotide sequence of the clone was confirmed by sequencing, the gbss A is 542bp and identified to 99.82% to the reported corresponding conserved sequence of GBSSⅠ(Genbank NO. X58453 ), the gbss B is 542bp and identified to 100% to the reported corresponding conserved sequence of GBSS. 2. The intron fragment of VP1-ABI3-like protein gene was amplified from potato DNA. The nucleotide sequence of the clone was confirmed by sequencing, the fragment is 237bp and identified to 100% to the reported corresponding intron sequence of VP1-ABI3-like protein gene(Genbank NO. AJ309218).3. Construction of the intermediate vector pSKgABI. The vector contains three sets of restriction sites: BamHⅠand PstⅠ, PstⅠand HindⅢ, XhoⅠand HindⅢ, they were used to splice and connect gbss A,intron and gbss B respectively.4. Connection of patatin promoter. The 35S promoter of plant expression vector was replaced by patatin and designated as pBI121g-P.5. Construction of the ihpRNAi plant expression vector -pBI121g-PgABI promoted by patatin promoter, then integrated pBI121g-PgABI into agrobaterium LBA4404 and engineering bacteria contains pBI121g-PgABI plasmid was obtained.6. The transgenic plants were obtained by transforming engineering bacteria into potatoes. The microtuber slice and stem segment of potato cultivar'Longshu 3'were used as plant material, six kinds of differentiation mediums of microtuber slice and stem segment were established by adding different hormones in MS medium. The microtuber slice of potato cultivar'Gannongshu 2'were used as plant material and the differentiation medium was MS+1.00mg/L IAA+0.20mg/L GA3+2.00mg/L ZT+ 0.50mg/L 6-BA.The results showed that: there were 36.5% differentiation rate and 61% rooting rate of resistant shoot of'Gannongshu 2'. The microtuber slice and stem segment of potato cultivar'Longshu 3'became flocculent and fluffy, so we did not obtain resistant shoot of'Longshu 3'.