Gene Transformation Of Sedoheptulose-1,7-bisphosphatase (Msbpase) In Mulberry

SBPase is a key enzyme in Calvin cycle process which catalyzes the reaction in the calvin cycle in the balance between assimilation and regeneration. SBPase also control the flow of carbon cycle in calvin cycle process. Previous studies on gene function of SBPase and found that it have improved the photosynthetic performance. Starch and soluble sugar content were increased in leaves of transgenic, what ever, the plant growth rate and dry matter accumulation were significantly increased, these all indicating a good prospect.Mulberry genetic engineering is not only an important part of mulberry breeding, but also an important basic research of silkworm industry. At present, for the types of purpose gene transformation used in mulberry is small, and was based on resistance gene. There is no report at home and abroad about improve the quality of mulberry leaves, or increase the leaf yield. Consider the case, this paper establish sterile system and the leaf regeneration system with mulberry buds as explants, analyzes and compares the differences in different mulberry species, and select agrobacterium-positive strains by optimizing the genetic transformation of leaf disc each factor, carried out mulberry SBPase Gene (MSBPase) Genetic transformation, a stable and efficient genetic transformation system, transgenic resistance mulberry buds ultimately. The main results are as follows:1 Impaction of buds of different bits, different species, different seasons were discussed on the initiation of culture, and the faction of different varieties effected on the subculture was analysised, the test results showed that: In the start training process, the effect of mulberry varieties, the drawing season and bud position on start have reached a significant level: Press the start rate, the most from fast to slow in the order of: XINSANG 11> XINSANG 13> FENGTIAN 2> HAINING> FENGTIAN 6> FENGTIAN 5; The spring buds is better than the autumn buds; the Ministry bud is the best, followed by the central bud and the lower part of shoots: the best start medium for Mulberry is MS +6- BA 2.00 mg/L +2,4-D 0.10 mg/L. In subculture, the affected of varieties, hormone type and ratio on growth and differentiation of mulberry is significantly: The merits of mulberry varieties in subculture is XINSANG 11> XINSANG 13> FENGTIAN 2> FENGTIAN 6> FENGTIAN 5> HAINING SANG; 6-BA ,2,4-D is the greatest impact on the growth and differentiation of mulberry; Impact of 6-BA is up to a significant level, NAA is not conducive to growth and differentiation of mulberry; The best medium for the differentiation is MS +6- BA 2.00 mg/L +2,4-D 0.10 mg/L, the mulberry value-added coefficient of 4.6 and the growth height is 4.8cm on the 30th day.2 Factors that affect leaf regeneration by orthogonal test, leaf browning inhibitors were screened,and using the single-factor test to determine the rooting medium of mulberry, test results showed that: The effect of mulberry variety, leaf position, hormone combination on callus induction rate of leaf were significant levels: The merits of the order of the tested varieties of XINSANG 11> NONGSANG 14> XINSANG 13> GUISANGYOU 12 ; The 6th leaf callus induction rate is better than the other leaves; The best medium for callus induction MS +6- BA1.00 mg/L +2,4-D 0.80 mg/L is getted with Duncans'multiple comparisons; XINSANG 11 Leaf callus induction rate is 95%. In the differentiation medium MS +6- BA 3.0 mg/L + IAA 0.1 mg/L, the XINSANG 11 of adventitious buds rate is up to 87%, the average shoot number is up to 5.47; Additional PVP 2.0 g/L, the lowest rate of leaf browning is 3%; XINSANG 11 root number is up to 5.86 in rooting medium 1/2MS + NAA 0.2 mg/L.3 With leaves as tissue culture'XINSANG-11'as explant, Single factor test to determine the genetic transformation of the concentration of the best infection, infection time, a total culture time, kan screening concentration, cef inhibitory concentration combinations..The mulberry leaf disc transformation system was established at last, experiments show that: the best concentration of bacterium infection is OD600=0.3; the time that 10 mins'infected of mulberry leaves drive to be appropriate; co-cultured 2 d was the most beneficial transformation; to first screening is 20 mg/L, the concentration of Kan in secondary screening is 30 mg/L , add 400 mg/L of Cef for inhibition at the same time,will not only get a higher transformation frequency of shoot regeneration, but also a better inhibitory effect. This transformation system conversion efficiency is 10%.4 Polyacrylamide gel electrophoresis showed that MSBPase gene has been imported into the mulberry, mulberry gene transfer of MSBPase initial success.