Studies On In Vitro Development Of Lilium Bulblet And Enzymes Related To Its Starch Synthesis

The edible lily bulblet is slow to develop,and it usually takes a long time to cultivate the bulblet formed by scale from the basal leaf seedling to the seedling with main stem in the process of planting and production.In this process,the bulblet changed from small to large,the starch content of bulblet accumulated gradually,this process is also the process of starch enrichment.From the point of view of starch biosynthesis metabolism,the developmental physiology of bulblet,the regulation mechanism of starch synthase genes related to bulblet formation and enlargement were studied in order to understand the regulatory function of key genes and the internal and external factors related to the development of bulblet.The establishment of efficient bulblet cultivation technology system has positive guiding significance for bulblet production in lily crop farming.In the research of lily（Lilium davidii var.unicolor（Hoog）Cotton）bulblet development by tissue culture technology,according to the growth characteristics of lily,which is easy to sprout cluster bud and difficult to grow bulblet,this study set up four culture stages,namely,cluster bud proliferation stage,small bulblet induction stage,bulblet swelling growth stage and the stage of swelling bulblet differentiate main stem.Different culture schemes were screened in each stage in order to achieve the optimization of technology.Observation of starch granule accumulation during bulblet development of lily by electron microscopy,and determination of starch content and activities of starch synthase AGPase GBSS and SSS in bulblets developed at different stages.In order to study the expression of the related enzyme genes,the AGP,GBSS,SSS gene was cloned,and fluorescence quantitative PCR was used to analyze the expression of AGP GBSS and SSS genes in the four stages of bulblet development.In order to study the regulation of starch synthesis metabolism during the development of lily bulblet by starch synthesis rate-limiting enzyme gene AGP,thus affecting the bulblet development.A 300bp conserved sequence of AGP gene was selected as interference fragment,and gateway technique was used to construct RNAi vector that interfered with mRNA transcript of AGP gene,and reverse down-regulated expression was carried out by genetic transformation.Fluorescence quantitative PCR was used to detect the relative expression of AGP in transformed plants,Western blot was used to detect the protein expression of AGPase in the transformed plants.The main results are as follows.1.The combination of 0.3 mg/L BA and 0.03 mg/L NAA could promote the stable subculture and proliferation of the cluster buds of lily tissue culture seedling,and the multiplication coefficient of the buds was determined at cluster bud proliferation stage.At the stage of bulblet induction and development,the lower part of cluster bud（bottom of leaf）metamorphosed and developed by culture with increasing sucrose concentration,along with the layers of scale formed from inside to outside,small bulblet formed finally.At the stage of bulblet swelling,the combination of 0.15 mg/L BA and 0.15 mg/L GA₃ and high concentration of sucrose were used to make the bulblet grow and swelling rapidly.This stage is the most important step in the whole culture process.the stage of main stem differentiation and development from the swelling bulblet is to continue to culture the bulblet to start to differentiate the main stem from the growing point of the bulblet accompanied by differentiating the inner scale.At this stage,the transformation from basal leaf seedling to stem seedling was completed.The formation and development of bulblet were observed by electron microscope at different growth stages of tissue culture bulblet,and it was found that the starch granules at the base and inside of the bulblet increased sharply.At the same time,the differentiation and formation of the main stem at the growing point of the bulblet disc were observed,which indicated that the bulblet with the main stem differentiation could be effectively cultivated by the four-stage culture plan.2.The starch content of lily bulblet in the four stages increased gradually,which was19.96%,32.54%,42.68%and 46.52%,respectively,and reached the significant level.AGPase enzyme activity increased significantly in the first three stages,and remained stable in the 4th stage.In the 4th stage,the main stem was differentiated,and the sucrose distribution was transferred to the bulblet for starch synthesis,which was also used for the morphogenesis of stem differentiation.The activity of GBSS is similar to that of AGPase,but the activity of SSS is the lowest in the third stage.The change of starch content and the activity of starch synthesis limiting enzyme AGPase were consistent with the changes of starch granule accumulation and bulblet enlargement observed by electron microscope.3.The AGP,GBSS,SSS genes were cloned and the sequences were submitted to the NCBI,getting the accession number KP751443,KP751445,and KP751444.By bioinformatics analysis,the cloned AGP sequence has 918bp,containing an open reading frame of 867bp,encoding 289 amino acids.Its amino acid sequence is a conserved domain of"PLN02241"with adenosine transfer catalytic function,the 8-184 amino acids belong to the conserved domain of"Glyco_tranf_GTA_type"and has the function of glycosyl transfer catalysis.The 208-289 amino acids belong to the conserved domain of"LbetaH",which contains five turns,has acyltransferase activity.The whole coding region is involved in the metabolism of starch and sugar.The GBSS sequence has 567bp,encoding189aminoacids,andthe3-188aminoacidsbelongtothe"Glycosyltransferase_GTB_type"domain,which has the function of glycosyl transfer and the catalytic activity of starch synthesis.The SSS sequence has 1257bp,encoding 419amino acids,belong to the"GT1_Glycogen_synthase_DULL1_like"domain which is related to catalyzing the formation of theα-1,4-glycoside bond."ADP-binding pocket"feature was formed at the 1,4,244-246,305-306,311,328,333 amino acid and"homodimer interface"feature was formed at the 172,348,353-354,356,388 amino acid.It has the function of binding to the substrate.According to the structure analysis of three main enzyme genes associated with starch synthesis,it shows that the three genes play an important role in starch synthesis and metabolism in the process of lily bulblet formation and development.The results of fluorescence quantitative PCR test showed that the expression of the three genes increased significantly with the development of bulblet,and the expression in bulblet was significantly higher than that in leaf.4.This study established the prokaryotic expression vector of AGPase and the polyclonal antibody was prepared for detection the specific reaction with lily AGPase protein.The constructed prokaryotic expression vector pET28-AGPase expressed 34kDa protein detected by SDS-PAGE.By Western blot test,the antibody serum can react with antigen protein specifically.The selected interference sequence was inserted into the entry vector pDONR221 by BP reaction using Gateway technique,with that,the sequence was inserted into the interference vector pJawohl8-RNAi by LR reaction.it showed that pDONR221-AGP entry clone and pJawohl8-RNAi-AGP expression clone were successfully constructed.The transgenic plants with down-regulated expression of AGPase protein were induced by genetic transformation of lily callus,indicating that the conserved sequence of AGP could be used as interference fragment to down-regulate mRNA transcription of endogenous AGP in lily.It provides information for studying the effect of AGP gene on starch biosynthesis and regulation of bulblet development in lily.