Study On The Function Of Sucrose Synthase Gene During Bulblet Development In Lilium.

As one of the four international cut flowers,Lilium plays an important role in the global flower industry.Bulblets production is a key link in the Lilium industry.Existing research shows that bulblet development is closely related to starch accumulation.As the main form of sugar transport in Lilium bulblets,sucrose is involved in starch synthesis under the regulation of sucrose synthase,which is of great significance to the formation of bulblet morphology.At present,domestic and foreign researches on bulblet metabolism during Lilium plant growth and development are limited to physiological level,and molecular research is still scarce.In order to explore the regulation mechanism of SuSy on sucrose-starch metabolism of Lilium bulblets,based on the successful cloning of LdSuSy gene in the early stage,this experiment systematically explored the molecular physiological function of LdSuSy gene.For the first time,using CRISPR / Cas9 gene editing technology,the Lilium LdSuSy gene was successfully edited to obtain a knockout mutant;Simultaneously construct LdSuSy overexpression vector and use Agrobacterium-mediated method to obtain transgenic lines;Using knockout mutants and LdSuSy transgenic lines as materials,combined with q RT-PCR technology and physiological measurements,the effects of LdSuSy on Lilium plants and bulblet development were studied.Here are some major experiment results:1.We successfully established usion expression vectors LdSuSy1-GFP and LdSuSy3-GFP fusion expression vectors.Transient transformation in tobacco leaves,by laser confocal observation,it was confirmed that LdSuSy1 and LdSuSy3 were localized to the plasma membrane.2.We successfully established LdSuSy overexpression vector p RI101-LdSuSy1 and p RI101-LdSuSy3 by double digestion;and established LdSuSy targeting vectors p HUE411-LdSuSy1 and p HUE411-LdSuSy3 by CRISPR/Cas9 gene editing technology.After Agrobacterium-mediated transformation of sterile scales of Lilium longiflorum ‘White Heaven',we screened three of LdSuSy1 overexpression lines,three of LdSuSy3 overexpression lines by PCR and real-time quantitative PCR;and screened two of Ldsusy1 knockout mutation plants,two of Ldsusy3 knockout mutation plants by PCR and sequencing.3.The growth and development indexes of Lilium transgenic lines of LdSuSy were observed and measured.The results show that under the same culture conditions,both overexpression of LdSuSy1 and LdSuSy3 can promote the growth and development of Lilium plants.The specific manifestations are enlarged bulblets and broadened leaves.The Ldsusy1 and Ldsusy3 knockout mutation plants show different morphological characteristics,the leafarea of Ldsuys1 knockout mutation lines increase,while the root length of the Ldsusy3 knockout mutation lines was significantly reduced.4.The expression of LdSuSy,SuSy enzyme activity and carbohydrate content in the transgenic lines of Lilium were determined.The results show: in LdSuSy1 and LdSuSy3over-expressing lines,the expression of LdSuSy in bulblets increased significantly,the enzyme activity significantly increased,and the starch content also increased.In the knockout mutation plants of Ldsusy1 and Ldsusy3,SuSy enzyme activity in the bulblets decreased and starch,the starch content also decreased slightly.In summary,the LdSuSy gene of Lilium is an important gene that regulates Lilium bulblets development.Its up-regulated expression can significantly promote starch accumulation in Lilium bulblets and promote leaf development in the aerial parts.The knockout mutant bulblet and leaf development are significantly inhibited.The above results laid a theoretical foundation for clarifying the sucrose-starch metabolism mechanism of Lilium bulblets,and provided technical support for Lilium directed molecular breeding,high-quality bulb breeding and production.