Characterization Of Genes Encoding SuSy And INV Key Enzymes For Starch-Sucrose Metabolism In Lilium Davidii Var. Unicolor Using RNA-seq

Starch is the dominant form of stored carbohydrate in Lilium bulbs, while sucrose is the main form of transported sugars. Consequently, starch-sucrose metabolism is vital to bulb development. Typically, sucrose is degraded by either SuSy or INV into hexose or their derivatives, which are then used as materials for starch synthesis. Accordingly, it is of great importance to gain insight of the physiological and molecular function of SuSy and INV for making clear the pathway of starch synthesis. Aim to illuminate the mechanism of SuSy and INV regulate the sucrose taking part in starch synthesis, the differential expressed SuSy and INV genes were selected based on the transcriptome analysis of bulblets development stages, and their overexpression and RNAi vectors were then constructed. The main results were showed as below:1. An efficient and steady RNA extraction technique from different parts of bulbs has been established through modifications of three common RNA extraction methods, CTAB, SDS and TRIzol method. After modified, both SDS and CTAB methods can obtain high quality RNA with clear bands, the brightness of 28S band was about 2 times of 18S band, and the ratio of A260/A280 was between 1.8 to 2.2. However, TRIzol method was failed to get rid of the polysaccharides. The results of RT-PCR indicate that the improved CTAB method is suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction.2. We conducted comparative transcriptome profiling of bulblets at 0 d,15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation. Analysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 nt were generated, and N50 was 926 nt. Thorugh BLAST, 37,385 unigene were annotated, almost accout for 70%. Based on COG analysis,11,150 unigene successfully aligned were classified into 24 categories, and 8% of the sequences were attributed to carbohydrate transport and metabolism. According to SSR analysis,1596 SSR sites were obtained.3. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC,18S,60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ would be appropriate whereas ACT together with AP4, or ACT along with GAPDH would be suitable for normalization of leaves and scales in different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4 showed the most stable expression, respectively. This study provides a guide for the selection of a reference gene under different experimental conditions, and will be benefit for further gene expression studies in a wide variety of Lilium.4. Carbohydrate metabolism, especially starch and sucrose metabolism, is crucial to the Lilium genus since it is vital to the formation and development of bulblets. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative expression profiling of bulblets. The results showed that the most different expressed genes were gained in the stage 15 d, which was regard as the most active stage. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that SuSy and INV, mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while SPS showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction showed higher gene expression in mother scales than in bulblets.5. The overexpression vectors and RNAi vectors of SuSy genes-SuSyl, SuSy2, SuSy3 and INV gene SAI were constructed. Take advantage of Gateway thechnology, the overexpression vecor ppMDC-SuSy3 and RNAi vector pB7WIWG-SuSy3 were successfully constructed. Through Gibson Assembly, the overexpression vectors of SuSyl, SuSy2 and SAI pCAMBIA-S1, pCAMBIA-S2 and pCAMBIA-SA1 were constructed. The RNAi vectors pTCK-S7, pTCK-S2 and pTCK-SAI were constructed use double enzyme digestion.Finally, we identified the differently expressed SuSy and INV genes for starch-sucrose metabolism in Lilium bulbs formation and development through RNA-seq, then analyzed the expression pattern of these genes, and contructed their overpression and RNAi vectors, to lay foundation for clarifying the physiological action and gene function of SuSy and INV, elucidating the starch synthase pathway, and producing good bulbs.