【作者】 任海燕；

【导师】 杜俊杰；

【作者基本信息】 山西农业大学 ， 果树学， 2018， 博士

【摘要】 枣胚早期败育严重是制约枣杂交育种进程的重大难题,激素水平与胚胎发育有着密切联系,然而关于内源激素水平调控枣胚胎发育的机理尚不清楚。为了更好地从生理、分子生物学角度揭示枣果实发育过程中胚败育与内源激素变化的相互关系,本研究以中等败育品种‘冷白玉’枣为材料,在观察其胚发育和败育状况的基础上,分析了枣果发育过程中内源激素水平的变化规律,并利用高通量转录组测序分析与影响枣胚败育激素相关基因及其表达特性,得出同时进一步探索了‘冷白玉’枣幼胚培养技术。以期从生理和分子生物学角度揭示枣胚败育与果实发育过程中内源激素变化的相互关系,为枣种质创新尤其是杂交育种提供理论指导。主要研究结果如下:1、阐明了‘冷白玉’枣胚生长发育动态,明确了枣胚败育时期,表明该品种在花后20d、30d、40d、50d胚胎发育状态分别为原胚、小球形胚、球形胚、鱼雷形胚;花后40d（球形期阶段）出现败育高峰。2、分析了‘冷白玉’枣果发育过程中胚胎发育与内源激素含量变化的关系。种胚中IAA的含量显著高于果肉,果肉中GA₄的含量高于种胚。种胚中低水平的GA₄且较高水平的IAA,及后期低含量的tABA和cABA、高含量的IAA/GA₄有利于幼胚的正常发育。胚发育过程种IAA下降、tABA增加及（GA₄+IAA）/ABA比值下降是导致胚败育的重要因素。3、利用RNA-Seq建立了‘冷白玉’枣败育高峰期的正常胚与败育胚的转录数据库,筛选出1219个差异表达基因,在内源激素KEGG代谢途径中筛选出差异基因为16个,败育胚相对于正常胚下调基因为14个,上调基因2个。败育胚中AUX/IAA（生长素诱导蛋白基因）、SAUR1（生长素早期响应基因）、PYR/PYL（脱落酸受体蛋白基因）、ERF1/2（乙烯应答转录因子）、GH3（吲哚乙酸酰胺合成酶）、PR-1（病程相关蛋白基因）相对于正常胚下调表达量倍数大。4、采用RT-qPCR分析了10个重要激素相关基因,正常胚中GH3.17（吲哚乙酸酰胺合成酶基因3.17）、AUX22D（生长素诱导蛋白）的表达量显著高于败育胚。败育胚中PLY（脱落酸受体）、ERF1B（乙烯应答转录因子）、EBF1、EBF2（乙烯信号调控因子1、2）的表达量显著高于正常胚,说明这些基因上调表达活化了ABA和乙烯的生理活性,促使了枣胚的败育。败育胚的Aux/IAA18（生长素/吲哚乙酸蛋白基因）、SAUR1（生长素早期响应基因）、GH3.1、GH3.6（吲哚乙酸酰胺合成酶基因3.1、3.6）的表达量显著高于正常胚,说明枣胚败育进程中,生长素早期应答基因家族表达增强是对胚衰败、生长素相对过量而发生的应激响应。5、激素与枣胚发育密切相关,各激素间的平衡影响着幼胚发育。通过筛选、优化枣胚培养过程中激素配比,最终对‘冷白玉’30d幼胚培养获得了成功。适宜‘冷白玉’枣幼胚培养的最小胚龄为花后30d。胚龄20d、30d、40d的幼胚成胚率分别为4.98%、56.82%、62.31%。30d幼胚直接成苗率为0.41%。幼胚愈伤组织诱导率为68.70%。在24℃条件下幼胚分化率可达81.12%。在胚乳看护培养的基础上,可促使幼胚沿胚性生长发育成苗,而未能沿胚性生长的幼胚,选用器官发生型愈伤组织再生途径,获得完整植株。两种途径的结合,有效减少了胚性后代植株的损失,为枣育种技术开辟了新途径。更多还原

【Abstract】 The early abortion of jujube embryo is a serious problem which restricts the process of hybrid breeding of jujube.Although the embryonic development is closely related to the plant endogenous hormone levels,the mechanism of the embryonic development of jujube regulated by the endogenous hormone is not yet clear.Therefore,in order to better reveal the relationship between endogenous hormones and fruit development during the process of jujube embryo abortion,changes of endogenous hormone levels during the development of ‘Lengbaiyu’ jujube fruit were studied based on the field investigation and microscopic observation.In addition,high-throughput transcriptome sequencing was also used to analyze the genes related to embryo abortion hormone and its expression characteristics during ‘Lengbaiyu’ jujube embryo abortion and fruit development.‘Lengbaiyu’ jujube immature embryo culture technology was also studied at the same time.The results of this study will be useful in germplasm innovation,especially crossbreeding in jujube.The main results obtained are as follows:1.The developmental dynamics and the abortion stage of embryos in ‘Lengbaiyu’ jujube were clarified.The results showed that the original embryo,small globular embryo,globular embryo and torpedo embryo appeared at the 20 d,30d,40 d and 50 d after flowering,respectively.And the peak of embryos abortion appeared at 40 d after flowering(globular embryo stage).2.The relationship between embryo development and endogenous hormone content during jujube development showed that the GA content in the embryo was significantly lower than that in the pulp,but the IAA content was significantly higher than that in the pulp.In addition,the lower level of GA,higher level of IAA in the embryo,the lower levels of t ABA,c ABA and the high content of IAA/GA were beneficial to the normal development of immature embryos.The decrease of IAA in embryos,the continuous increase of t ABA,the ratio of(GA+IAA)/ABA in embryos and pulp were the important factorsthat leaded to embryo abortion.3.Based on RNA-Seq analysis,1219 differentially expressed genes were screened between normal and abortive embryos at the peak of abortive stage of ‘Lengbaiyu’ jujube,among which 16 differential genes were related to the endogenous hormone metabolism pathway.Among the 16 genes,14 genes were down-regulated and 2 genes were up-regulated.The expression of AUX/LAA(auxin-inducible protein gene),SAUR1(auxin early response gene),PYR/PYL(abscisic acid receptor protein gene),ERF1/2(ethylene-responsive transcription factor),GH3(indoleacetamide synthesis))and PR-1(pathogenesis-related protein gene)were down-regulated,and the expression of AHP(histidine phosphotransferase gene)were up-regulated.4.The RT-q PCR results showed that the expression levels of GH3.17(indole acetate synthase gene3.17),AUX22D(Auxin-induced protein)in normal embryos were significantly higher than those in abortive embryos.The expression levels of PLY(abscisic acid receptor),ERF1B(ethylene response transcription factor),EBF1,EBF2(ethylene signaling regulators 1,2)in abortive embryos were significantly higher than those in normal embryos.Therefore,we believed that these up-regulated genes could effectively activate the physiological activities of ABA and ethylene,which in turn promotes the abortion of jujube embryos.In addition,the gene expression level of Aux/IAA18(auxin/indole acetate protein gene),SAUR1(auxin early response gene),GH3.1,GH3.6(indole acetate synthase gene 3.1,3.6)in abortive embryos were also significantly higher than normal embryos.This is the stress response of the gene family in the early response of auxin,which is the feedback of the relative excess auxin in the abortive embryo.It shows that hormone regulatory genes resulting abortion,and embryo abortion causes changes of endogenous hormones in young embryos.5.Hormones are closely related to the development of jujube embryos,and the balance between hormones affects the development of immature embryos.By screening and optimizing the hormone ratio during the jujube embryo culture,the culture of ‘Lengbaiyu’ 30 d embryos was finally succeeded.The youngest embryo successfully-rescued was 30 days old.The percentage of immature embryos in20-day-old,30-day-old and 40-day-old could reach to 4.98%,56.82% and 62.31%.The plantlet formed rate of younger embryo in 30-day-old directly was 0.41%.The younger embryos induction ratio was 68.70%.With the culture temperature maintained 24℃,the differentiation rate with this formula was the highest,reached to 81.12% at 24 ℃.On the basis of endosperm care culture,the immature embryos could bepromoted to grow into embryos along the embryonic growth,and the immature embryos that fail to grow along the embryogenicity were selected from the organogenesis-type callus regeneration pathway to obtain intact plants.The combination of both methods of plant regeneration directly from immature embryos or from callus induction greatly improves the utilization of immature embryos for obtaining sufficient hybrid seedlings for jujube breeding.更多还原