| Starch is a renewable, macromolecule material. It not only plays a dominant role in food industry, but also increasingly be applied in the fields of materials, medicine, biology and so on. Because of the excellent properties such as higher water content, less quantity of protein residue, lower gelatinization temperature, stronger suction tension and better expansibility, etc, potato starch has already been important raw materials in food and industry applications.Amylopectin and amylose are two important components of starch. They are used in respective fields of application for the different molecular structures and the states of molecular gathering, Amylose's property is similar to fiber's, the film made from it has good transpapency, flexibility, tensile strength and water–insolublity and is nonpoisonous and nonpolluting. It can be widely used in the sealing materials, packaging materials, water-resistant materials, corrugated and adhesives industry. Amylopectin has the properties of aging resistance, high expansibility, water absorption, thickening effect and it can improve the freeze-thaw stability. It is widely used in many fields, such as food processing, packing materials, water-soluble and biodegradable films, medicine, architecture, etc. Therefore, it has great significance to cultivate high amylopectin or high amylose potato varieties.Granule-bound starch synthase is the enzyme which combines with starch granule and, directly related to the synthesis of amylose starch. In this study, ihpRNAi vector of potato GBSSI gene was transformed into microtuber slice of potato mediated by Agrobacterium tumefaciens. PCR and PCR-southern molecular-detection-methods were used to verify transgenic plants, then, the starch structure and related physiological indexed of tuber were analyzed to investigate the effect of ihRNAi vector on potato starch in order to obtain the new potato lines with high amylopectin content.The results have showed as follows:1. The microtuber slice of tetraploid potato cultivar"Gannongshu 2"was used as experimental material. The differentiation medium was MS+1.00 mg/L, IAA+0.20 mg/L, GA3+2.00 mg/L, ZT+0.50 mg/L, 6-BA medium. The results showed that: there were 36.5% differentiation rate and 61% rooting rate of resistant shoot of"Gannongshu 2". The nptⅡgene and target gene-ihpRNAi segment of GBSSI gene of transgenic plant, which were identified by Kan positive resistance ,were detected by using PCR method.The results showed that the expected segments of 61 transgenic plant were obtained in total 68 transformed plants., accounting for 90% of the chosen resistant plants. The PCR-southern detection result showed that PCR-amplified products were truly nptⅡgene and target gene, which proved that ihpRNAi fragment has been integrated into the genome of potato.2. The colour of starch granule treated with potassium iodide was observed using optical microscope. In transgenic potato, the amylopectin content is much higher than amylose, so the red or mixed red and blue colour of starch granule of transgenic potato were observed. However, in non-transgenic potato tubers, the starch granules were blue and red,and blue starch granule is more than red starch granule .It showed that the ihpRNAi segment limited the expression of GBSSI gene, correspondingly, the amount of amylose in transgenic plant tubers was decreased and the amount of amylopectin was increased remarkably.The amylopectin and amylase content of starch of the transgenic lines and control potato were measured using dual wavelength spectrophotometric method. In transgenic potato tubers, amylopectin content is notably higher than amylose. In transgenic potato tubers, the GBSS enzyme activities which control amylose were notably lower than that of non-transgenic plants, while the SSS enzyme activities controlling amylopectin were significantly higher than that of non-transgenic plants.3. Compared to non-transgenic potato tubers, reducing sugar, soluble sugar, soluble protein, dry matter, flour yield, etc, in transgenic potato tubers were not remarkably different. It indicated that the ihpRNAi segment of GBSSI gene had no effects on these physiological characteristics of potato tubers. |
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