黄瓜GLABROUS3（CsGL3）基因调控果刺发育的分子机制解析

Cucumber spine is a kind of typically trichome that cover the surface of the cucumber fruit. It is an important external quality trait, that affects the market value with respect to spine formation, for example, absence and presence. However, to date, the regulatory mechanisms underlying the development of fruit spine are largely unknown. In this study, CsGL3 was obtained with a homologous cloning method. This was consistent with the gene that was isolated by map-based cloning conducted by other researchers worldwide. Ectopic expression of CsGL3 could complement Arabidopsis gl2-8 mutant deficient in trichome. The result of PCsGL3-GUS expression indicated that CsGL3 may be involved in the regulation of spine (trichome) initiation. In suit hybridization analysis showed that CsGL3 was abundantly expressed in the epidermis, suggesting that CsGL3 may play an important role in epidermal cell differentiation. Based on the results we obtained, we will analyze the biological function of the target gene as well as the relationship between CsGL3 and IAA. In addition, to explore the interaction between CsGL3 and CsGL1 or other genes which could be involved in the regulation of spine development. Furthermore, to provide important insights into the role of CsGL3 in the regulatory network regulating fruit spine and trichome formation of cucumber. We hope the results presented here will be helpful to reveal more about the genetic regulatory mechanism of cucumber spine, and to enrich and deepen the cell differentiation theory for cultivated plants.

黄瓜果刺是果实表皮上凸起的一种表皮毛，属果实重要商品性状，且果刺的发育状况是果实商品性状主要构成因素，但目前人们对黄瓜果刺发育的分子机制及其调控途径尚知之甚少。课题组利用同源克隆的方法已经获得了与国内外最近通过图位克隆定位一致的GLABROUS3(CsGL3)基因。异位表达试验表明，该基因可成功互补拟南芥无毛突变体gl2-8的表型；PCsGL3-GUS表达结果表明，CsGL3可能参与了果刺的起始；原位杂交结果则进一步说明CsGL3在表皮细胞优势表达，暗示该基因可能在表皮细胞分化中具有重要作用。本研究拟利用课题组获得的csgl3和csgl1突变体材料，解析CsGL3基因生物学功能及其与生长素的关系，探究CsGL3与CsGL1及其它参与果刺调控基因的互作，深入阐释CsGL3参与黄瓜果刺及表皮毛形成的分子调控途径。研究结果将有助于揭示黄瓜果刺的遗传调控机制，丰富和深化栽培植物细胞分化理论。

果刺是黄瓜果实表皮凸起的一类特殊表皮毛。果刺的发育状况是影响果实商品品质的关键性状，但有关果刺发育的分子机制及调控途径尚不明晰。针对上述存在的科学问题，为探明果刺发育中一些重要基因的功能及调控机理。课题组利用获得的一份彻底无毛的突变材料（csgl3），开展了CsGL3的生物功能研究；解析了CsGL3与信号因子之间的作用、CsGL3与CsGL1以及其他未知调控基因的相互作用。取得的重要结果如下：1）明确了CsGL3的表达模式及亚细胞定位：CsGL3主要在果实皮层表达且CsGL3定位于细胞核；2）阐明了CsGL3在果刺起始中的作用：利用CRISPR-Cas9基因编辑技术明确了CsGL3的突变可导致黄瓜植株彻底无毛；3）发现CsGL3与生长素调控表皮发育的过程无关：利用外源喷施生长素不能恢复csgl3的表型，csgl3转录组数据中生长素转导相关基因表达无显著差异，CsGL3对生长素处理无响应；4）解析了CsGL3与CsGL1及其它未知调控基因的相互作用：遗传学试验证实了CsGL3与CsGL1存在隐性上位作用，利用单突及双突转录组数据，鉴定了91个NAC基因，其中12个可能参与了果刺的形态建成；发现位于CsGL3下游的MIXTA-like MYB类转录因子MYB6表达模式与CsGL3表达类似，且与经典的TRY存在互作，MYB6-TRY功能验证表明该模块在调控果刺发育过程发挥重要功能。综上，该研究证实了CsGL3的生物学生功能，建立了CsGL3-CsGL1-MYB6-TRY为核心的调控模块。研究结果不仅丰富了调控果刺发育的基因资源，同时也为以果刺为目标的黄瓜分子育种奠定了理论基础，实践及科学意义重大。