| ‘Fuji’ is the main cultivars of apple in China, accounting for more than 65% of the total cultivated area. However, ‘Fuji’ have a long period of children, and poor quality of flower bud, and alternate bearing in which seriously restricting the development of China’s apple industry. At present, shoot bending as an important measure to promote the ‘Fuji’ apple flower bud formation has been widely used, but the gene expression regulation network of apple flower bud induction and molecular regulation mechanism is not clear. Therefore, it is beneficial to enrich the basic theory of flower bud differentiation of fruit trees by analysis of gene expression pattern and molecular regulation mechanism in response to shoot bending,and it has an important theoretical and practical significance for developing the regulation technology of apple flower bud formation, and genetic improvement of apple flowering characteristics. In this paper, we use ‘Nagafu No. 2’ and ‘Qinguan’ as materials to analys gene expression pattern of apple flower bud induction by RNA-seq technology, and compare the two varieties of flowering related genes DNA polymorphism variation by re-sequencing technology, and clarify the gene expression pattern of flower bud induction, and the role of mi RNAs in flower bud induction in response to shoot bending, as well as in vegetative phase transition of fruit trees. Combining the gene expression pattern of flower bud induction and bioinformatics, we study the functionof apple Md IDD transcription factor family genes in flower induction, so as to provide a theoretical basis for the genetic improvement of apple varieties of early flowering characteristics. The main results are described as follows:1. Construction the buds RNA-seq libraies of ‘Nagafu No. 2’ during physiological differentiation stage and analysis of differentlly expression genes patterns, and determination of carbohydrate content and metabolism related enzyme activity, and hormone level in buds and leaves.(1) In the early stages of flower buds induction, the higher sucrose content and starch accumulation were necessary condition for flower induction in apple. And expression profiles of sugar related genes showed a consistent change trend with them.(2) In the early stages of flower induction, the peak of IAA and GA content appear more earlier, the more easier to flowering. Before the flower bud induction, the peak of CK content reached the earlier, the more easier to flowering, and these hormone related genes showed a consistentchange trend with them.(3) Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds,indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. However, changes in sugar and starch deposition in buds can be affected by ABA levels and the expression levels of genes involved in the ABA signaling pathway. In a word, the hypothetical model for the regulation of flower induction in apple by sugar and hormone signaling crosstalk was proposed.2. Based on re sequencing technology, we re-sequenced two elite apple varieties ‘Nagafu No.2’ and ‘Qinguan’, with different characteristics. And analysis and comparison of DNA polymorphism between two varieties.(1) We identified many genome variations, including2,771,129 and 2,262,888 single nucleotide polymorphisms(SNPs), 82,663 and 63,764 structural variations(SVs), and 1,572,803 and 1,294,060 insertion/deletions(INDELs) in‘Nagafu No.2’ and ‘Qinguan’, respectively.(2) 171,520 and 147,090 non-synonymous SNPs spanning 23,111 and 21,400 genes, and 3,963 and 3,196 SVs in 3,431 and 2,815 genes, as well as 1834 and 1451 INDELs in 1681 and 1345 genes were observed for ‘Nagafu No.2’ and‘Qinguan’, respectively.(3) Genetic linkage maps of 190 flowering genes associated with multiple flowering pathways in ‘Nagafu No.2’, ‘Qinguan’, and ‘Golden Delicious’ was established and these genes were used for analyzing DNA polymorphisms among three apple varieties in which were closely related to phenotypic traits.3. Construction buds RNA-seq libraies of shoot bending and control treatments in‘Nagafu No. 2’ apple trees during physiological differentiation stages and the differentially expressed genes that involved in sugar metabolism, hormones, stress response and flowering were identified in the treatments of shoot bending and control.(1) The rate of bud breaking and flowering, and proportion of spur were significantly increased in response to shoot bending.(2) The levels of carbon metabolism and sugar contents were changed, and expression levels of sugar metabolism related genes were significantly up-regulated, and accelerated the transfer of sugar molecules signal and induced flower bud formation in response to shoot bending.(3) Biosynthesis and signal transduction of ABA, ET, SA and JA related genes were significantly up-regulated, as well as WRKY transcription factor and R genes also showed up-regulation of expression patternsin response to shoot bending.(4)Flowering related genes were significantly up-regulated expression in response to shoot bending.4. Construction buds mi RNAs libraies of shoot bending and control treatments in ‘Nagafu No. 2’ apple trees during physiological differentiation stages and identification of differentially expressed mi RNAs.(1) 188 mi RNAs belonging to 39 families and 137 novelmi RNAs were identified. And 68 and 27 mi RNAs were down and up expression in response to shoot-bending, respectively, and there were 31 differently expression novel mi RNAs.(2) In addition, hormone related mi RNAs and their targets including mi R396, 160, 159, 319, and164, as well as GRFs, ARFs, MYBs, TCP4, and NAC1 targets showed significantly differently expression in response to shoot-bending.(3) Some important flowering control genes(SPLs,SOC1, FT, AFL1, and AP1) also were significantly up regulation expression in response to shoot-bending. In a word, the model of shoots-bending promote flower buds formation by hormone related mi RNAs mediated regulation in apple was proposed.5. In this study, M. hupehensis mi RNAs and degradome libraries, in which were adult phase leaf for mi RNAs and juvenile phase leaf for mi RNAs and degradome libraries, were constructed. And differently expression mi RNAs were identified between them.(1) We identified 42 known mi RNA families and 172 novel mi RNAs.(2) We also identified 127 targets for 25 known mi RNA families and 168 targets for 35 unique novel mi RNAs.(3) The model of molecular regulation mechanism of phase transition by mi R156-SPLs, mi R172-AP2 and auxin related mi R160 and mi R393 and their target genes mediated regulation in fruit trees was proposed.6. In this study, twenty Md IDD family genes were identified in Malus domestica Borkh.,and they contains four highly conserved zine finger domain including ZF1(CCHH), ZF1(CCHH), ZF1(CCHC) and ZF1(CCHC).(1) The expression profiles of 10 Md IDD genes in‘Nagafu No. 2’ and ‘Yanfu No.6’, as well as in response to sucrose treatment were measured using q RT-PCR. The expression of 10 Md IDD genes were significantly higher in ‘Yanfu No.6’ buds and 8 Md IDD genes were significantly higher expression in response to sucrose than in control treatments.(2) In addition, the c DNA sequence of seven Md IDD genes were isolated from ‘Nagafu No. 2’ buds using RT-PCR.(3) Contruction of the plant expression vector p VBG2307/Md IDD2 and selection of Md IDD2 T1 Arabidopsis. |
PDF Full Text Request