蓝莓花粉直感效应调控花青素合成的生理与分子机制研究

Blueberry is the fruit tree species with self-incompatible or low self-compatiblity that needs urgent attention in cultivation. It is the effective approach to resolve the problem that planting with cultivars combination, but there is a significant influence on the intrinsic quality of anthocyanin, and it is closely related to the paternal characteristics which is the typical metaxenia phenomenon. However, so far, how to regulate anthocyanin synthesis by metaxenia has not been clearly explained. Therefore, on the basis of prophase research work, the physiological mechanism of the regulation on anthocyanin synthesis by metaxenia is systematically researched from five aspects with the idea of physiological processes of metaxenia. Including differences in pollen viability and content of endogenous hormones and polyamines in pollen of different species, pollination compatibility of different pollination combinations, content of endogenous polyamines and hormones of style before and after pollination, content of endogenous polyamines and hormones of the flesh and seeds of young fruit at different stages of development among different pollination combinations. Simultaneously, the molecular mechanism of the regulation on anthocyanin synthesis by metaxenia is systematically researched by transcriptomics of four life organisms and different developmental stages, including the pollen of different species, the style of before and after pollination of different pollination combinations, the flesh and seeds of young fruit at different stages of development among different pollination combinations. The purpose of this study is to reveal the critical step of the regulation on anthocyanin synthesis by metaxenia and the gene expression characteristic of differentially expressed genes, to explore the biological pathway of differentially expressed genes, to expound the physiological and molecular mechanism of anthocyanin synthesis by metaxenia, to provide scientific basis for better understanding of metaxenia, directive breeding, regulation of anthocyanin biosynthesis, and artificial control of fruit quality.

蓝莓绝大多数品种自交不亲和或自交亲和性低，生产中需多品种搭配种植以提高坐果率，以解决这一难题，但对花青素等果实内在品质亦有显著影响，且与父本有着密切的联系，表现出典型的花粉直感效应。然而，迄今为止，花粉直感效应如何调控花青素合成仍未获明确解释。因此，在前期研究的基础上，本项目以花粉直感效应产生的生命过程为线索，拟从不同品种花粉活力、内源激素与多胺差异，授粉亲和性，授粉前后花柱和幼果(果实)中内源多胺和激素的变化和差异，系统地分析花粉直感效应调控花青素合成的生理机理。同时从不同品种花粉，不同品种花粉授粉前后的花柱、不同发育阶段幼果的果肉和种子的转录组系统地分析花粉直感效应调控花青素合成的分子机制。揭示花粉直感效应调控花青素合成的关键步骤，差异表达基因的表达特性及生物学通路，阐明花粉直感效应调控花青素合成的生理和分子机制，为定向育种，调控蓝莓花青素生物合成、深入认识花粉直感现象提供科学依据。

花粉直感效应在蓝莓中普遍存在，然而其如何调控花青素合成仍未获明确解释。本研究已花粉直感效应产生的生命过程为线索系统地分析了花粉直感效应调控花青素合成的生理机理和分子机制。结果表明，蓝莓花粉活力与花粉中IAA、GA3、ZT和Cad含量呈极显著正相关，与ABA含量及ABA/IAA、ABA/GA3、ABA/ZT和ABA/(IAA+GA3 +ZT)之值呈极显著负相关。低含量IAA、GA3和ZT，及高含量ABA积累，(IAA+GA3+ZT)/ABA之值相对低，ABA/GA3、ABA/ZT之值高则不利于花粉管生长，表现出自交不亲和性。‘杰兔’自花和异花授粉后果实中花青素含量与6-BA、ZT、CTK、和ABA含量，及(CTK+6-BA+ZT)/GA3比值呈极显著正相关；与IAA、NAA含量，及(IAA+IPA+NAA)/(CTK+6-BA+ZT)、(IAA+IPA+NAA)/ABA和ABA/(CTK+6-BA+ZT)比值呈极显著负相关。授粉后第80 d为花青素合成的关键时期，通过转录组分析，筛选了调控花青素合成通路中“花青素3-O-葡糖基转移酶”的2个差异表达miRNAs 及1个靶基因。筛选了调控类黄酮合成通路中“查尔酮异构酶”的差异表达的novel_miR_41及3个靶基因，调控“根皮苷合酶”的候选差异表达的novel_miR_767及1个靶基因，调控“黄烷酮还原酶4”和“黄烷酮醇还原酶4”的9个差异表达miRNAs及2个靶基因。筛选了调控苯丙素生物合成通路中“肉桂酰辅酶A还原酶”的3个差异表达miRNAs及5个靶基因，调控“β-葡萄糖苷水解酶”的7个差异表达miRNAs及11个靶基因，调控“咖啡基莽草酯酶”的差异表达novel_miR_911及3个靶基因，调控“过氧化物酶”的5个差异表达miRNAs及3个靶基因，调控“咖啡因o -甲基转移酶”的差异表达novel_miR_840及1个靶基因，调控“松柏基乙醛脱氢酶”的差异表达novel_miR_83及1个靶基因。综合分析表明，内源激素，差异表达miRNA及靶基因在苯丙素、类黄酮和花青素合成途径中调控一系列催化酶基因的差异表达，这在调控花青素生物合成中发挥关键作用。研究结果为明确花粉直感效应调控花青素合成的关键时期和差异表达基因的调控通路，阐明花粉直感效应调控花青素合成的生理和分子机制提供科学依据，具有重要理论价值和实践意义。