苹果IAA代谢几个关键基因在矮化砧木与miRNA在短枝品种中致矮作用研究

发布时间：2018-01-19 05:13

本文关键词： 苹果生长素矮化砧木短枝型　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：苹果矮化密植栽培是世界苹果发展的趋势,实现苹果矮化栽培最主要途径是利用好矮化砧木及短枝型品种。虽然矮化砧木及短枝型品种在生产中普遍应用,但矮化机理,特别是分子机理尚未完全揭示。本文分析了长富2号/M9(矮化砧木)和长富2号/MM106(半乔化砧木)生长素代谢相关基因的表达情况,研究了几个关键基因在致矮中的功能和作用。利用miRNA组测序手段,分析了普通型富士长富2号和短枝芽变烟富6号中枝条发育相关差异miRNA。从生长素和miRNA角度,揭示矮化砧木和短枝型品种的致矮机理,丰富苹果矮化调控理论。取得的主要研究结果如下:1、对长富2号/M9和长富2号/MM106叶片、接穗韧皮部、砧木韧皮部和根系中的生长素含量、生长素合成基因、生长素运输基因和生长素代谢相关基因进行了分析,结果发现,长富2号/M9中生长素的浓度低于长富2号/MM106。长富2号/M9砧木韧皮部的生长素浓度高于其接穗。在长富2号/M9叶片和根系中,生长素合成基因MdYUCCA8和MdYUCCA10a表达显著低于长富2号/MM106。长富2号/M9砧木韧皮部和根系中,生长素运输基因MdPIN1b和Md PIN8a显著低于长富2号/MM106。长富2号/M9中的生长素轭合酶基因MdGH3-5b和MdGH3-9a显著低于长富2号/MM106。然而,长富2号/M9中的生长素水解酶基因MdIAR3c和MdILL6c显著高于长富2号/MM106。2、在M9和MM106中克隆了MdYUCCA8基因,该基因编码1272 bp核苷酸,二者序列相似性99.9%,编码423个氨基酸。克隆了MdYUCCA8编码区上游1600 bp启动子序列,二者相似性为99.6%。MdYUCCA8启动子受到GA、MJ和Br诱导,亚精胺和乙烯利显著抑制了MdYUCCA8启动子活性。高温促进MdYUCCA8的表达,烟草叶片双荧光素酶瞬时表达实验证明MdPIF4a与MdYUCCA8启动子互作。在拟南芥中异位表达MdYUCCA8,促进了拟南芥植株花茎的生长、顶端优势显著增加、侧枝减少、角果畸形、促进了根系的生长,侧根数量和侧根长度显著高于野生型。3、在M9和MM106中克隆了MdYUCCA10a基因,该基因编码1149 bp核苷酸,二者序列相似性99.9%,编码382个氨基酸。克隆了MdYUCCA10a编码区上游1800 bp启动子序列,二者相似性为99.7%。MdYUCCA10a启动子受到ABA、Spe诱导,乙烯利显著抑制MdYUCCA10a启动子活性。在拟南芥中异位表达MdYUCCA10a,促进了拟南芥植株花茎的生长,促进了根系的生长,主根长度、侧根数量和侧根长度显著高于野生型。4、在M9和MM106中克隆了MdABCB19基因,其编码1250个氨基酸,含有两个ABC_membrance结构域,两个ABC_tran结构域。该序列在M9和MM106中存在一个非同义SNP,编码氨基酸由A突变为S,导致M9α螺旋少了一段。MdABCB19在砧木M9和MM106、长富2号/M9和长富2号/MM106均差异表达。启动子序列分析发现M9的MdABCB19启动子在起始密码子上游170 bp处有“CTCTGT”6个碱基缺失,导致M9缺失了一个5’UTR Py-rich stretch motif。MM106 MdABCB19的启动子活性高于M9 MdABCB19,并且MdABCB19启动子受光照调控。MdABCB19启动子活性随着5’UTR Py-rich stretch motif元件数目的增加而增强。5、在长富2号及短枝型芽变烟富6号顶梢中,共发现700个成熟的miRNA,包括202个苹果已知mi RNA和498新miRNA。长富2号和烟富6号中有135个差异表达的miRNA,大多数差异的已知miRNA在烟富6号中下调表达。烟富6号枝条顶梢IAA、GA、ZR和ABA含量都低于长富2号。喷施GA可以促进烟富6号短枝生长,变为长枝。烟富6号顶梢中细胞伸长和细胞分裂相关基因的表达水平都显著低于长富2号。综上所述,生长素在砧木诱导接穗矮化中起着重要作用,长富2号/M9中低表达的生长素合成基因MdYUCCA8和MdYUCCA10a可能导致长富2号/M9中低的生长素含量。M9 MdABCB19启动子5’UTR Py-rich stretch motif缺失,可能造成M9中MdABCB19表达量降低,生长素运输减弱,植株矮小。GA和miRNA在烟富6号短枝发育中起着关键调控作用。
[Abstract]:The dwarf and close planting apple is the world trend of development, the realization of Apple dwarfing cultivation the main way is to use a good rootstock and spur type. Although the dwarf rootstock and spur type which is widely used in the production, but the Dwarfing mechanism, especially the molecular mechanism has not been fully revealed. This paper analyzes the /M9 (dwarf Nagafu No. 2 stock) and /MM106 (Joe Nagafu No. 2 half of the stock) the expression of auxin metabolism related genes, the study of several key genes in the function and role of dwarfing in group miRNA. The sequencing methods, analyzes the common type of Fuji Nagafu No. 2 and No. 6 short branch bud smoke rich in shoot development related differences from miRNA. auxin and miRNA angle, reveals the Dwarfing mechanism of Dwarfing Rootstock and spur type variety, rich apple dwarf control theory. The main results are as follows: 1, the Nagafu No. 2 /M9 and Nagafu No. 2 leaf /MM106, scion toughness Firstly, the content of IAA in root and rootstock phloem, auxin biosynthesis gene, auxin transport genes and auxin metabolism related genes were analyzed. The results showed that auxin concentration of auxin Nagafu No. 2 in /M9 is lower than that of Nagafu No. 2 /MM106. Nagafu No. 2 /M9 rootstock phloem is higher than that of the scion. In Nagafu No. 2 /M9 leaves and roots, expression of auxin synthesis genes MdYUCCA8 and MdYUCCA10a were significantly lower than the Nagafu No. 2 /MM106. Nagafu No. 2 /M9 rootstock phloem and root, auxin transport genes MdPIN1b and Md were significantly lower than that of PIN8a / MM106. yoke Nagafu No. 2 auxin Nagafu No. 2 in /M9 synthase gene MdGH3-5b and MdGH3-9a were significantly lower than the Nagafu No. 2 /MM106. however. Nagafu No. 2 in /M9 MdIAR3c and MdILL6c were significantly higher than that of auxin hydrolase gene Nagafu No. 2 in /MM106.2, M9 and MM106 in MdYUCCA8 gene was cloned and the gene encoding the 1272 BP core The nucleotide sequence similarity of two, 99.9%, encoding 423 amino acids. The cloning of the MdYUCCA8 encoding region 1600 bp upstream promoter sequence, two similarity to 99.6%.MdYUCCA8 promoter by GA, MJ and Br induced by spermidine and ethephon significantly inhibited the MdYUCCA8 promoter activity. The expression of MdYUCCA8 in high temperature to promote the tobacco the leaves of dual luciferase transient expression experiments demonstrated that MdPIF4a and MdYUCCA8 promoter interactions. Ectopic expression of MdYUCCA8 in Arabidopsis thaliana, promote Arabidopsis stem growth, apical dominance increased significantly, branches reduced, pod deformity, promote root growth, root number and root length were significantly higher than that of the wild type.3, M9 and MM106 in clone the MdYUCCA10a gene, the gene encoding 1149 BP nucleotides, two sequence similarity 99.9%, encoding 382 amino acids. The cloning of the MdYUCCA10a encoding region 1800 bp upstream promoter sequence, two similarity For the 99.7%.MdYUCCA10a promoter by ABA, induced by Spe, ethephon significantly inhibited the activity of MdYUCCA10a promoter. Ectopic expression of MdYUCCA10a in Arabidopsis thaliana, promote Arabidopsis stem growth, promote root growth, root length, root number and root length were significantly higher than those in the wild type.4, M9 and MM106 in MdABCB19 gene was cloned. The encoding 1250 amino acids, containing two ABC_membrance domains and two ABC_tran domains. The sequence has a non synonymous SNP in M9 and MM106, encoding amino acid substitution from A to S, resulting in M9 alpha helix is less of a.MdABCB19 in stock M9 and MM106, the expression of /M9 and Fuji Nagafu No. 2 2 No. /MM106 were different. The promoter sequence analysis indicated that the M9 promoter of MdABCB19 "CTCTGT" 6 nucleotide deletion in the upstream of the start codon 170 BP, resulting in a loss of M9 5 UTR Py-rich stretch motif.MM106 MdAB " The CB19 promoter activity was higher than that of M9 MdABCB19, and MdABCB19 promoter by the light regulation of.MdABCB19 promoter activity increased with 5 UTR Py-rich stretch motif "the number of components and enhanced.5, in Nagafu No. 2 and spur type sports smoke rich 6 tops, 700 were found in mature miRNA, including 202 Apple MI is known RNA and 498 new miRNA. Nagafu No. 2 and No. 6 in smoke rich 135 differential expression of miRNA, miRNA is known most differences in tobacco rich down regulates the expression of 6. 6 branches tops smoke rich IAA, GA, ZR and ABA content was lower than that of Nagafu No. 2. Application of GA can promote smoke into the rich 6 short branches grow into long branches. The expression level of tobacco rich cell elongation and cell division related genes in 6 tops are significantly lower than those of Nagafu No. 2. In summary, auxin induced scion plays an important role in Dwarfing Rootstock, low expression of Nagafu No. 2 /M9 synthase gene M auxin DYUCCA8 and MdYUCCA10a may cause Nagafu No. 2 /M9.M9 low auxin MdABCB19 promoter 5 'UTR Py-rich stretch motif deletion, may cause decreased MdABCB19 expression in M9, auxin transport decreased, plant small.GA and miRNA rich 6 short branch development plays a key role in the smoke.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S661.1

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