| Birch (Betula platyphylla Suk.) is an important species of afforestation which is one of the main tree species distributions in northeast and Inner Mongolia. Because of the importance of ecological, ornamental and practical economic value in birch, the scope of genetic improvement of birch has gradually expanded in currently. The main purpose is cultivating fast-growing, high-quality, high photosynthetic efficiency and high resistance birch variety. Therefore, the variety creation of high photosynthetic efficiency and fast-growing birch based on the natural birch using molecular biology methods, is not only providing a theoretical basis for forest tree molecular biology research, but also can obtain high quality birch in the economy value aspect to provide material for creation of new varieties of trees. In this study, we choose BpMYB106 overexpression line and non-transgenic B. platyphylla as materials, which gene is related development of trichome, analyze spatio-temporal expression of BpMYB106, trichome density, photosynthesis, transpiration rate, and growth rate, meanwhile combine with expression profile for analysis of the downstream genes. Therefore, these results provide reference to reveal pleiotropy function of BpMYB106, which is a novel MYB gene in the role of photosynthetic rate, growth of birch and trichome density,. Thus we can make some reference for screening of high efficiency, high growth rate of birch quality varieties.1. The sequence of BpMYB 106 was cloned from birch, which was belonged to R2R3 MYB gene family. BpMYB 106 sequence was similar to a series of MYB genes which were related to regulation of photosynthesis and trichome development from Arabidopsis (Arabidopsis thaliand), poplar (Populus trichocarpa) and maize (Zea mays). Therefore BpMYB 106 was speculated that it was associated with photosynthesis and trichome development.2. The expression pattern of BpMYB 106 in different tissues of the wild-type plants was analyzed, and the results showed that the expression level of BpMYB106 in shoot tip and leaf were significantly higher than stem and root. This result indicated that BpMYB 106 exhibited a tissue specific expression in shoot tip and leaf.3. pBI121-BpMYB106-GFP fusion vector was constructed to transient transfomate onion epidermal cell by gene gun method, and the result showed BpMYB 106 was localized in cell nuclei.4. The upstream sequence 1573 bp of BpMYB 106 promotor was cloned by Genome Walking. The analysis of cis-acting element showed that promotor contained many photosynthetic, chlorophyll and MYB binding sites. The 1500 bp sequence of promotor was cloned into pBI121-GUS vector instead CaMV35S promotor to construct the GUS fusion vector pMYB-GUS. Transient infection in birch showed that the BpMYB106 promoter exhibited activities in new leaves (shoot tip) and trichomes of old leave.5. We constructed pROK Ⅱ-BpMYB106 overexpression vector for birch transformation and eleven transgenic lines were obtained. Overexpression of BpMYB106 caused increased trichome density. These lines had an obviously higher plant height. In BpMYB106 overexpression lines, photosynthetic rate and transpiration rate were higher than wild birch. However, the quantity of stoma and water loss rate were found no significant difference (P>0.05).6. The expression profile showed 992 differential genes in overexpression birch, which contained 508 upregulated genes and 484 downregulated genes. There were 12 differencial expression genes related to photosynthesis and oxidative phosphorylation in expression profile. Besides, genes that correlated with glutaredoxin, plant-pathogen interaction, auxin, phenylpropanoid biosynthesis and flavonoid biosynthesis were included in the differential genes.7. The analysis of promotor cis-acting element of photosynthesis, oxidative phosphorylation, auxin related genes showed that every sequence contained MYB biding sites. Besides, photosynthesis genes promotors had rbcS consensus sequence and Ibox/Ibox core, oxidative phosphorylation genes promotors had T box, SORLIPs and Box Ⅱ elements, auxin genes promotors have ARF element. The analysis of flavonoid biosynthesis pathway showed six upregulated genes which respectively encoding flavonoid 3’-monooxygenase (F3’H), flavonoid 3’,5’-hydroxylase (F3’5’H), bifunctional dihydroflavonol 4-reductase (DRF), leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), one downregulated gene which encoding chalcone synthase (CHS), and these enzymes were key enzymes for flavonoid biosynthesis. p-Coumaroyl-CoA was substrate of flavonoid biosynthesis. Four genes encoding enzymes for synthesis of p-Coumaroyl-CoA also differentially expressed, they were phenylalanine ammonia-lyase (PAL) and 4-coumarate--CoA ligase (4CL).8. cis-acting elements from 12 differencial expression genes which related to photosynthesis and oxidative phosphorylation in expression profile were chosen to construct pHIS2-element vector, which elements names were MYB1AT, MYB2AT, MYBCORE, MYBST1, MYBZM, SORLIP2, Box Ⅱ and RBCS. Meanwhile, pGADT7-Rec2-BpMYB106 vector was constructed. We exploited yeast one-hybrid system for Y187 cotransformation. The cotransformated yeasts which correlated with the elements mentioned above were grow up. The results indicated BpMYB106 could interact with those MYB and light-responsive elements in the promoters of 12 BpMYB106-activated photosynthesis and oxidative phosphorylation genes. |
PDF Full Text Request