Study On The Establishment Of Optimized Transformation System And Introduction AGPase Gene Into Cassava

Cassava is one of three big yams crops in the world and is a valuable source ofcalories for about 600 million people in the developing tropical countries where fooddeficiency and malnutrition are often common. As one of the most important energyproduced root tuber crops in the tropic, Cassava is defined as an ideal feed stock forproduction of renewable fuel ethanol by Chinese government. It is a question how toenhance starch yield in cassava tuberous root. AGPase plays a critical role in the regulationof starch synthesis in plants, not only because it catalyses the first dedicated step in starchsynthesis, but also because it is the rate-limiting step in starch Synthesis.Antisense-mediated inhibition of AGPase expression has been shown to lead to a severedecrease in starch production. So production enhanced of ADP-glucose would increasecassava tuberous root starch production as a result of cassava's enhanced capabilities toproduce photosynthate.In this paper, as a template of genomic DNA of E. coli JM109, glgC gene wasamplified by polymerase chain reaction (PCR). The nucleotide 1007 was changed from Gto A via site-directed mutagenesis, and the amino acid was changed from glycine 336 toaspartic acid accordingly. As a result of prokaryotic expression, the SDS-PAGE analysisindicated that the recombinant protein was 53kDa which was the same to the reportedarticle. The expression level of its protein was about 77.3% of the total protein of theinduced recombinant bacteria at the ,presence of IPTG .Moreover, the expression level ofthe site-directed mutated gene, named glgC336, was higher than glgC.glgC336 gene was constructed into a plant expression vector named pCB-C336 andthus it was made to express itself in transgenic tobacco plants. The results indicated theconstruction of the plant expression vector was successful, which was proved by GUS genewith histochemical method and molecule detection, and the starch content in the transgenictobacco had been increased 13.1%, compared to Non-transgenie tobacco. This gave asufficient and essential preparation to transform cassava main cultivars, for the purpose ofincreasing their starch content. The late result indicated expression of the glgC336 gene inall tissues led to weakly performing,plants, presumably, as a result of improper allocation of carbohydrate between phototrophic and heterotrophic tissues. So this vector was notused in cassava transformation.The two high effective regeneration systems were obtained by the comparison ofdifferent regeneration system by analyzing the different effects. The result showed thatsomatic embryos induced rate of 'SC 8' is highest and lowest of 'SC 7' among 6 recipientmaterials. Somatic embryos induction medium: MS+0.5mg/L CuSO₄+4 mg/L 2,4-D.The best adventitious buds induction medium: MS+0.5 mg/L CuSO₄+1.0 mg/L 6-BA+0.5 mg/L IBA+6 mg/LAgNO₃. Highly efficient plant regeneration via germination ofsomatic embryos was achieved using maltose instead of sucrose.Before the cassava transformation, effects of the explants growth state, Kan sensibility,the period of pre-culture, bacterial infection and co-cultivation on the transgenic efficiencywere investigated. The result showed that the best explants were growing on embryomaturation medium for 2 weeks. The concentration of kanamycin was 15mg/L for the stageof adventitious buds induction and 10mg/L for root formation. Highly GUS transientexpression rate was achieved by the following parameters: OD₆₀₀ 0.4 of GV3101 bacterialliquid, 30～45 minutes of infection and 3～4 days of co-cultivation. 6cm of bombardmentdistance and one time could obtain the highest GUS transient expression rate for particlebombardment.Based on the optimized transformation system, the expression vectors pSP-C336containing glgC336 gene under the control of root tuberous specific expression promoter(Sporamin) were used in the cassava transformation. Transgenic cassava plants wereobtained by Agrobacterium-mediated transformation and particle bombardment. PCR andSouthern blot analysis showed that glgC336 gene has been integrated into PCR andSouthern blot analysis showed that glgC336 gene has been integrated into cassava genome.RT-PCR and Northern blot analysis demonstrated that glgC336 gene has expressed attranscriptional level in the transgenic cassava. It is also evidenced that the AGPase activityand starch content in transgenic cassava plants was increased by 35.54% and 1.59 percentrespectively.