Background

Rubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield.

Objective

To understand the biological function of the GhRCAβ2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAβ2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern.

Methods

The bioinformatics tools were used to analyze the sequence features of GhRCAβ2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAβ2 protein. The expression pattern of the GhRCAβ2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR).

Results

The full-length CDS of GhRCAβ2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAβ2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAβ2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAβ2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA β-isoform. The GhRCAβ2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAβ2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAβ2 in comparison to the wild-type cotton plants. The GhRCAβ2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAβ2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAβ2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCAβ2 gene.

Conclusion

Our findings will establish a basis for further understanding the function of the GhRCAβ2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances.

中文翻译：

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陆地棉 (Gossypium hirsutum L.) Rubisco 激活酶基因 GhRCAβ2 的分子特征和表达模式

背景

Rubisco激活酶（RCA）是碳同化途径中催化Rubisco激活的关键酶。许多研究表明，RCA 可能是旨在提高光合作用效率和作物产量的基因操纵的潜在目标。

客观的

为了了解陆地棉GhRCAβ2基因的生物学功能，我们克隆了GhRCAβ2基因的编码序列（CDS），并研究了其序列特征、进化关系、亚细胞定位、启动子序列和表达模式。

方法

利用生物信息学工具分析GhRCAβ2蛋白的序列特征。对拟南芥叶肉原生质体进行瞬时转化以确定 GhRCAβ2 蛋白的亚细胞定位。通过分析转录组数据并使用定量实时 PCR (qRT-PCR) 检查GhRCAβ2基因的表达模式。

结果

GhRCAβ2的CDS全长为1317 bp，编码带有叶绿体转运肽的蛋白质。 GhRCAβ2 具有两个保守的 ATP 结合结构域，并且不具有植物中 RCA α 同工型特有的 C 末端延伸 (CTE) 结构域。进化上，GhRCAβ2 聚集在 A 组，与大豆 RCA 具有密切的进化关系。蛋白质印迹分析表明，GhRCAβ2 对 RCA 抗体具有免疫反应性，其分子量与 RCA β-亚型相似。 GhRCAβ2 蛋白存在于叶绿体中，与其在光合作用过程中作为重要酶的作用相符。GhRCAβ2基因具有叶片组织特异性表达模式，与野生型棉花植株相比，黄绿叶突变体表现出GhRCAβ2表达降低。 GhRCAβ2启动子含有多个对光、植物激素和应激作出反应的顺式作用元件，表明GhRCAβ2的表达可能受到这些因素的调节。对胁迫反应的额外检查表明GhRCAβ2表达受到冷、热、盐和干旱胁迫的影响。值得注意的是，在不同的应激条件下观察到不同的表达模式。此外，低磷和低钾胁迫可能导致GhRCAβ2基因表达显着降低。

结论

我们的研究结果将为进一步了解GhRCAβ2基因的功能奠定基础，并为在具有挑战性的环境条件下提高棉花光合效率和产量提供宝贵的遗传知识。