黑果枸杞响应土壤干旱胁迫的蔗糖分配对茎刺发育启动的影响及其机理

Lycium ruthenicum Murr. is regarded as an excellent eco-economical tree species for the control, development and utilization of both sandy land and saline-alkali land. The stem thorns of L. ruthenicum belong to the induced defense system. However, the corresponding thornless L. ruthenicum is more suitable for cultivation as an economic tree. We have found that the formation of L. ruthenicum stem thorns is enhanced by soil drought stress, and suppressed by both the higher supply of sucrose and higher level of soil moisture. The higher supply of sucrose is the initial regulator of branches development from axillary buds. Also, stem thorns are developed from axillary buds. Thus, we have speculated that soil drought stress may reduce the supply of sucrose in axillary buds of L. ruthenicum to initiate stem thorn development. To verify this hypothesis, we will investigate both the contents of sucrose and glucose in axillary buds of L. ruthenicum plants, which suffer from soil drought stress (multiple thorn group) or not (thornless group), to reveal the correlation between sucrose allocation and initiation of stem thorn development. Moreover, we will reduce the sucrose content of the thornless group and increase the content of sucrose or its non-metabolizable analogues of the multiple thorn group to reveal whether the sucrose supply is signal for initiation of stem thorn development. Furthermore, transcriptome analyses, real-time PCR and RNA in situ hybridization will be carried out to uncover key genes involved in sucrose and stem thorn initiation and to reveal detailed temporal-spatial expression characters of the key genes. The objective of this project is to elucidate the influence and mechanism of sucrose allocation response to soil drought stress on initiation of stem thorns development. This provides foundation for breeding thornless L. ruthenicum.

黑果枸杞（黑杞）是用于治理和开发利用沙化土地及盐碱地的优良生态经济型树种。虽然黑杞茎刺属于诱导防御范畴，但是同等条件的无刺类型更宜作经济型林木栽培。我们发现土壤干旱胁迫促进黑杞茎刺形成，较高土壤水分和蔗糖供应均能抑制其茎刺产生。因为较高蔗糖供应是启动植物腋芽向侧枝发育的第一调节因子，且茎刺亦由腋芽发育而来。所以我们推测土壤干旱胁迫通过降低黑杞腋芽蔗糖分配来启动茎刺发育。为了验证这一假说，本项目拟通过分析土壤干旱胁迫多刺组和非胁迫无刺组黑杞腋芽蔗糖和葡萄糖含量来揭示蔗糖分配与茎刺启动的相关性，通过降低无刺组蔗糖含量和增加多刺组蔗糖或非代谢性蔗糖类似物含量来揭示蔗糖供应是否为启动茎刺发育的信号，通过转录组测序、实时荧光定量PCR和RNA原位杂交来揭示与蔗糖启动茎刺相关的关键基因及其精细时空表达特性。本项目旨在揭示响应土壤干旱胁迫的蔗糖分配对黑杞茎刺发育启动的影响及机理，为培育无刺黑杞奠定基础。

黑果枸杞为优良生态经济型灌木。黑果枸杞棘刺为日常管理带来诸多不便，同等条件的无刺类型更宜栽培。在发现高蔗糖抑制黑果枸杞刺发生的基础之上，我们推测响应干旱胁迫的低蔗糖供应对刺发育启动有非常重要的影响。为验证以上假说，本项目获得了体细胞无性系变异频率低的黑果枸杞无性系，摸索了抑制黑果枸杞组培无性系刺发生的方法，研究了黑果枸杞刺发育过程，分析了蔗糖及葡萄糖含量与刺发育启动的相关性，筛选了与蔗糖影响刺启动相关的候选基因，克隆了候选基因——蔗糖合酶基因（SUS）的cDNA全长。项目主要研究结果表明：（1）叶外植体的直接器官发生能力受内源IBA，而不是外源植物生长调节剂影响；茎外植体腋芽发生植株的变异频率最低，可为本项目提供遗传背景一致的无性系；（2）黑果枸杞刺起源于腋芽，为茎刺；刺形成后其基部仍有潜藏腋芽存在，且有“芽缝”与外界相通；（3）黑果枸杞刺启动期（Init）的蔗糖含量极显著低于无刺期（CK）和刺形成期（Form），而其葡萄糖含量则显著高于CK和Form；推测低蔗糖供应为茎刺启动的信号，高葡萄糖为茎刺启动提供能量；（4）“Starch and sucrose metabolism”KEGG路径的SUS基因，在三种材料（CK，Init 和Form）的表达水平与蔗糖供应呈显著正相关，推测低蔗糖合酶介导低蔗糖信号启动黑果枸杞茎刺发育。项目研究结果为黑果枸杞分子育种及培育无刺类型奠定基础。项目主要培养硕士研究生5名，获得授权国家发明专利2项，发表论文3篇，其中SCI收录论文1篇。