Study On NnGBSS Regulating Amylose Biosynthesis In Nelumbo Nucifera Gaertn

Lotus(Nelumbo nucifera Gaertn.)is a perennial aquatic herb of the lotus family,and is the largest cultivated aquatic vegetable in China.The expanded rhizome of lotus root is the main edible organ,and starch is the main component of the rhizome.The starch content of lotus root rhizomes accounts for more than 70%of the total dry matter mass of rhizomes.The content of amylose plays an important role in affecting the gelation,gelatinization and aging of starch molecules,and GBSS is the only key enzyme that is active during the synthesis of amylose.How does GBSS in lotus root regulate the synthesis of amylose?What are the differences in the structure and expression of GBSS among lotus root resources?What are the interacting proteins and transcription factors that affect the expression and function of NnGBSS?These issues are not yet clear.Therefore,to explore the effects of the structure,promoters,interaction proteins and transcription factors of NnGBSS on amylose synthesis will be effective in realizing the efficient regulation of lotus root rhizome starch quality and cultivating excellent lotus roots.The main research results are as follows:1.The relationship between NnGBSS expression and amylose contentUsing ’meirenhong’ and ’Z9’ as the test materials,it was found that with the development of lotus root rhizomes,the content of total starch and amylose also increased.There is a positive correlation between the content of amylose and the expression of NnGBSS.The higher the expression of NnGBSS,the higher the content of amylose.However,there are significant differences between the two materials.The amylose content and NnGBSS expression of’meirenhong’ in each period are much higher than that of’Z9’,and the greater the difference in amylose content in the later stage,the NnGBSS expression of ’meirenhong’ reached 5.2 times of ’Z9’.2.Comparison of the structure of NnGBSSThere are 5 differences in the amino acid sequence of NnGBSS between ’meirenhong’ and’Z9’.After PHYRE2 protein tertiary structure simulation,it is found that the alanine-threonine difference at position 382 results in less ’Z9’ than ’meirenhong’ an alpha helix that maintains the stability of the protein structure.The NnGBSS promoter is quite conservative.The sequence of the 27 tested materials is exactly the same,and there are elements related to growth regulators,photoperiod,temperature,and stress resistance.It was found that the NnGBSS protein was located on the cell membrane and cell nucleus by constructing the pGWB5-GBSS binary expression vector,and then transforming the Agrobacterium into the tobacco leaves.3.The upstream and downstream regulatory factors of NnGBSSBy constructing pGBKT7-GBSS vector for yeast two-hybrid screening library,EF1α,which interacts with NnGBSS,was isolated and identified.Its main function is to participate in protein synthesis,and it is also closely related to plant resistance to stress.The yeast one-hybrid screening library was carried out with a bait vector of 1500 bp upstream of the NnGBSS promoter,and three transcription factors of SAMS2,PUB 17 and SMT2 were obtained by BLAST comparison.They play a role in protein modification,salt,and oxygen stress.