黄瓜（Cucumis sativus L.）是设施栽培的主栽蔬菜之一，种植面积大，产量高，在保供“菜篮子”中发挥重要作用。目前，我国设施黄瓜种植面积已超过 128 万 hm²，在我国居民膳食中的比重达 18.5%，但黄瓜对高温的适应能力相对较弱，高温天气通常会影响黄瓜的生长和发育，特别是在设施栽培中，晚春和早秋常常伴有高温发生，导致叶绿（Chlorophyll, Chl）含量降低，植株早衰，严重影响产量和品质。Chl 是植物光合作用的关键色素，不仅能够捕获光能，而且将光能转化为化学能，在维持植物生长和发育方面起着不可替代的作用。Chl 含量的高低取决于合成和降解。在我国，特别是夏季和季节交换期间，设施栽培环境中的高温经常对黄瓜的生长发育造成不利影响，导致生理代谢紊乱，光合速率降低，甚至引发植株过早衰老，严重制约了黄瓜的高效生产。因此，我们需要探究高温对黄瓜叶片 Chl 合成的阻碍机制，以便有效缓解高温胁迫对黄瓜叶片老化过程的影响。这项研究不仅在理论上具有重要意义，而且对实际生产具有指导作用。本研究以热敏感黄瓜品种‘中农 12 号’为材料，明确高温诱导 Chl 合成通路关键酶基因 CsGluTR1，并通过遗传转化和基因沉默，获得 CsGluTR1 烟草过表达植株和黄瓜沉默株系，研究 CsGluTR1 在 Chl 合成中的功能。同时，利用酵母单杂交和双荧光素酶鉴定到 CsMYB44 与 CsGluTR1 启动子互作，研究了 CsMYB44 参与 Chl 的合成机制，并构建了高温诱导黄瓜叶片 Chl 合成受阻的分子途径，从转录调控角度阐明其分子机制，为黄瓜抗热栽培提供理论依据。主要研究结果如下：

1. 高温胁迫导致黄瓜植株生长缓慢，Chl 含量降低。Chl 合成酶基因 CsGluTR1、CsALAD 在高温胁迫下显著下调，编码 GluTR、ALAD 酶活性显著降低。谷氨酰-tRNA还原酶（Glutamyl-trna reductase, GluTR）催化生成的 5-氨基乙酰丙酸（5-aminolevulinic acid, ALA）含量在处理 12 d 时显著下降，而其合成前体物质谷氨酸（Glutamic acid, Glu）含量在处理 12 d 时显著上升。这些结果表明，高温诱导黄瓜叶片 Chl 合成受阻，受阻发生在 Glu 至 ALA 的合成阶段。

2. CsGluTR1 正向调控黄瓜叶片 Chl 合成。黄瓜基因组数据中存在两个 GluTR基因，该基因编码 552 个氨基酸，分子量 60.9 Kda 与拟南芥、烟草高度同源。CsGluTR1 在叶片中的表达量最高且响应高温、低温、干旱、黑暗等逆境胁迫及脱落酸（ABA）、 水杨酸（SA）、茉莉酸甲酯（MeJA）、赤霉素（GA3）激素处理的诱导。通过氨基个 GluTR 成员，并且它们均具有保守的结构域。高温条件下，过表达的 CsGluTR1 烟草株系中 Chl 含量、PSII 最大量子产量（Fv/Fm）、PSII 实际量子产量[Y（Ⅱ）]、净光合速率（Pn）、谷氨酸（Glu）以及 5-氨基乙酰丙酸（ALA）含量均显著升高；沉默 CsGluTR1 的黄瓜幼苗 Chl、ALA 含量降低，Glu 含量升高，表明 CsGluTR1 在高温 诱导 Chl 合成受阻中发挥关键作用，并且正向调控 Chl 合成。

3. 高温下 CsMYB44 通过抑制 GluTR1 表达导致 Chl 合成受阻。高温诱导CsMYB44 的表达，通过酵母单杂交、双荧光素酶试验发现 MYB44 能够结合高温诱导Chl 合成受阻关键位点 CsGluTR1 的启动子并抑制 CsGluTR1 的表达。与对照相比，高温下过表达的 CsMYB44 烟草植株 Chl 含量、ALA 含量、GluTR1 表达量显著下降，而Glu 含量显著上升。此外，高温下 MYB44 沉默黄瓜植株，ALA 含量、GluTR1 表达量显著上升，而 Glu 含量显著下降。表明 MYB44 通过负调控 GluTR1 转录水平，减少ALA 合成，导致高温下黄瓜叶片 Chl 合成受阻。

4. 高温诱导 CsMYB44 的 H3K9ac 水平升高。通过染色质免疫共沉淀技术和实时荧光定量 PCR（ChIP-qPCR）试验发现，高温下 CsMYB44 的 H3K9ac 水平显著升高。用去乙酰化抑制剂曲古柳菌素（Trichostatin A, TSA）处理黄瓜植株后，CsMYB44 的表达量显著提高。高温处理 3 d 后 TSA 黄瓜植株的 Chl 含量显著下降。上述结果表明，高温胁迫下 CsMYB44 的 H3K9 位点乙酰化水平升高，致使 CsMYB44 的转录水平升高，进一步导致黄瓜幼苗叶片 Chl 含量降低。

综上所述，CsMYB44 在高温诱导黄瓜叶片 Chl 合成中发挥关键作用。高温下 H3K9ac 促进了 CsMYB44 的表达，而 CsMYB44 的上调又进一步抑制了 CsGluTR1 的 表达，从而导致 Chl 合成过程中 Glu 到 ALA 位点发生阻碍，致使 Chl 含量降低。

Cucumbers （Cucumis sativus L.） is one of the main varieties of facility vegetables, with large planting area and high yield, and plays an important role in the supply "basket". At present, China's facility cucumber planting area has more than 2 million hm₂, the proportion of 18.5% in the diet of Chinese residents, but the ability of cucumber to adapt to high temperature is relatively weak, high temperature weather usually affects the growth and development of cucumber, especially in the facility cultivation, late spring and early autumn often accompanied by high temperature. The content of Chlorophyll （Chl） was decreased, and the plant had premature aging, which seriously affected the yield and quality.

Chl is a key pigment in plant photosynthesis, which not only captures light energy but also converts it into chemical energy, playing an irreplaceable role in maintaining plant growth and development.The level of Chl content depends on synthesis and degradation. In China, especially during summer and seasonal exchange, high temperatures in facility cultivation environments often adversely affect the growth and development of cucumber, leading to physiological and metabolic disorders, decreasing photosynthetic rate, and even triggering premature plant senescence, which seriously restricts the efficient production of cucumber. Therefore, we need to investigate the mechanism of high temperature obstruction to Chl synthesis in cucumber leaves in order to effectively alleviate the effects of high temperature stress on the aging process of cucumber leaves. This study is not only theoretically important, but also has a guiding role in practical production. In this study, we used the heat-sensitive cucumber variety 'Zhongnong 12' as the material, clarified the key enzyme gene CsGluTR1 of high temperature-induced Chl synthesis pathway, and obtained CsGluTR1 tobacco overexpression plants and cucumber silencing lines through genetic transformation and gene silencing to study the CsGluTR1 in Chl synthesis function in Chl synthesis. At the same time, we identified CsMYB44 interacting with CsGluTR1 promoter by using yeast single hybridization and dual luciferase, studied the mechanism of CsMYB44 involved in Chl synthesis, and constructed the molecular pathway of high temperature-induced blockage of Chl synthesis in cucumber leaves, elucidated the molecular mechanism from the perspective of transcriptional regulation, and provided the theoretical basis for the heat-resistant cultivation of cucumber. The main research results are as follows:

1. High-temperature stress resulted in slow growth and reduced Chl content in cucumber plants.The Chl synthase genes CsGluTR1 and CsALAD were significantly down-regulated under high-temperature stress, and the activities of the encoded GluTR and ALAD enzymes were significantly reduced. The content of 5-aminolevulinic acid (ALA) catalyzed by Glutamyl-tRNA reductase (GluTR) decreased significantly at 12 d, while the content of its synthesis precursor Glutamic acid (Glu) increased significantly at 12 d. The content of Chl synthesizing enzyme CsGluTR1 and CsALAD was significantly down-regulated. The content of Glutamic acid (Glu), the precursor of its synthesis, increased significantly at 12 d. These results suggest that high temperature induced a blockage of Chl synthesis in cucumber leaves, and the blockage occurred at the stage of synthesis from Glu to ALA..

2. CsGluTR1 positively regulates chlorophyll synthesis in cucumber leaves. There are two GluTR genes in cucumber genome data, among which GluTR1 is a key gene that restricts chlorophyll synthesis, encoding 552 amino acids. The molecular weight of 60.9Kda is highly homologous with Arabidopsis tobacco. The expression level of CsGluTR1 was the highest in leaves, which was in response to stress such as high temperature, low temperature, drought and darkness, and induction by ABA, SA, MeJA and GA3 hormone treatments. According to amino acid sequence comparison, Arabidopsis, tobacco and cucumber all have two GluTR members, while barley and wheat have three GluTR members, and they all have conserved domains. Under high temperature conditions, chlorophyll content, Fv/Fm, Y（Ⅱ）, Pn, Glu and ALA contents in overexpressed CsGluTR1 tobacco lines were significantly increased, while chlorophyll and ALA contents were decreased and Glu contents were increased in cucumber seedlings with silted CsGluTR1. These results indicated that CsGluTR1 affected the photosynthesis of plants and played an important role in chlorophyll synthesis.

3. The expression of CsMYB44 was induced by high temperature. It was found by yeast single hybrid and double luciflucifase experiments that MYB44 could bind the promoter of CsGluTR1, a key site hindered by high temperature induced chlorophyll synthesis, and inhibit the expression of CsGluTR1. Compared with the control group, chlorophyll content, ALA content and GluTR1 expression of overexpressed CsMYB44 tobacco plants were significantly decreased under high temperature, while Glu content was significantly increased. In addition, when MYB44 silenced cucumber plants at high temperature, the content of ALA and the expression of GluTR1 were significantly increased, while the content of Glu was significantly decreased. These results indicated that MYB44 played a key regulatory role in the high-temperature induced chlorophyll synthesis obstruction. At high temperature, CsMYB44 inhibited the expression of GluTR1, which reduced the synthesis of ALA and led to the obstruction of chlorophyll synthesis.

4. High temperature induced increased H3K9ac level of CsMYB44. The ChIP-qpcr test showed that the H3K9ac level of CsMYB44 at high temperature was significantly higher than that of the control group. In addition, after treating cucumber plants with the deacetylation inhibitor Trichostatin A （TSA）, the expression of CsMYB44 was significantly increased, and the chlorophyll content of TSA cucumber plants treated with 3d high temperature was significantly decreased compared with the control group. The results showed that the acetylation level of H3K9 site of CsMYB44 increased under high temperature stress, which led to the increase of CsMYB44 transcription level, and further resulted in the decrease of chlorophyll content in cucumber seedlings.

In conclusion, CsMYB44 plays a key role in the chlorophyll synthesis of cucumber leaves induced by high temperature. At high temperature, H3K9ac promoted the expression of CsMYB44, and the up-regulation of CsMYB44 further inhibited the expression of CsGluTR1, which resulted in the obstruction of the position of Glu to ALA during chlorophyll synthesis.