Pan-genomic Comparison and Functional Analysis of DELLA Genes in Soybean

HU Mingyu; HAO Peiyuan; BAI Wenqin; WANG Zhongwei; ZHANG Jijun; DU Chengzhang

doi:10.13718/j.cnki.xdzk.2024.08.001

2024 Volume 46 Issue 8

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HU Mingyu, HAO Peiyuan, BAI Wenqin, et al. Pan-genomic Comparison and Functional Analysis of DELLA Genes in Soybean[J]. Journal of Southwest University Natural Science Edition, 2024, 46(8): 2-11. doi: 10.13718/j.cnki.xdzk.2024.08.001

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HU Mingyu, HAO Peiyuan, BAI Wenqin, et al. Pan-genomic Comparison and Functional Analysis of DELLA Genes in Soybean[J]. Journal of Southwest University Natural Science Edition, 2024, 46(8): 2-11. doi: 10.13718/j.cnki.xdzk.2024.08.001

Pan-genomic Comparison and Functional Analysis of DELLA Genes in Soybean

1.
Chongqing Aacademy of Aagricultural sciences, Chongqing 401329, China
2.
Key Laboratory of Eevaluation and Utilization for Special Crops Germplasm Resource in the Southwest Mountains, Ministry of Agriculture and Rural Affairs (Co-construction by the Ministry and Province), Chongqing 401329, China
3.
School of Aagriculture, Yunnan University, Kunming 650504, China

More Information

Corresponding author: DU Chengzhang
Received Date: 19/05/2024
Available Online: 20/08/2024
MSC: S565.1

Abstract

The DELLA mutants improved crop yield by planting at high density, playing critical roles in "Green Revolution". Study on DELLA genes of soybean is expected to create varieties that are adapt to dense planting to improve the yield of soybean. In this study, 232 DELLA genes are identified by pan-genomic analysis from 29 soybean germplasm resources, 19 of which appeared frameshift mutations and cannot encode products with DELLA protein signatures. The results show that each germplasm resource contains 8 DELLA genes, which are located on different chromosomes. The proteins of GmGAI1s and GmGAI2s have typical DELLA domains. While, the GmRGL2 proteins and GmRGL1 proteins from chromosome 10 were inconsistent with the DELLA, LExLE and TVHYNP motif of GmGAI1s and GmGAI2s. The GmRGL1 proteins from chromosome 20 was defective in DELLA domain. The expression patterns show that GmGAI1s were expressed at a higher level in plant tissues and organs, may play a major role. The preferential expression of GmGAI2s, GmRGL1s and GmRGL2s in developmental flowers, pods and seeds, respectively, suggests that the DELLA genes function on the development of these tissues and organs. In addition, the interacting protein of soybean DELLAs was predicted, which provides a target on the downstream genes. Generally, these results are available for further clarifying the biological function of DELLA genes in soybean.
- soybean,
- DELLA genes,
- pan-genomic analysis

References

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大豆(Glycine max)是豆科大豆属一年生草本植物，其籽粒是植物蛋白和油脂的重要来源. 我国是大豆原产国，具有丰富的种质资源，但大豆消费严重依赖进口. 提高种质资源利用效率，增加大豆产量，对于促进大豆相关产业可持续发展有重要意义. 在“绿色革命”中，利用水稻和小麦矮生资源选育半矮化品种，提高种植密度，使作物产量急剧增加^[1]. 其中，水稻矮生资源sd1是赤霉素(Gibberellins，GAs)合成酶基因GA20ox的缺失突变体^[2]；小麦矮生资源rht1是DELLA基因功能获得性突变体^[1]，这两种突变体均促进了DELLA蛋白的累积，使株高降低. 因此，DELLA基因家族有重要的应用价值.

DELLA蛋白是GAs信号传导途径中的一类负调控因子，其N端具有DELLA结构域，包含DELLA，LExLE，TVHYNP 3段保守序列，是GAs受体蛋白GID1(Gibberelin Insensitive Dwarf1)的识别位点^[3-4]；C端具有GRAS蛋白家族的特征，包含LR1，NLS，VHIID，LR2，SH2-like，PFYRE，RVER和SAW等保守区，主要与顺式调控元件结合和其他调控因子互作. 在GAs的信号传导途径中，当GAs与GID1结合后，GID1的构象改变，进入细胞核作用于DELLA蛋白^{[3, 5-6]}. GAs-GID1-DELLA复合物与SCF E3泛素连接酶作用，使DELLA蛋白通过泛素化途径降解，启动下游基因表达^[7]. 当GAs浓度很低时，DELLA蛋白在细胞中累积，植物表现出矮化等性状^[8].

在拟南芥中发现了5个DELLA基因，即GAI，RGA和RGL1/2/3^[8-11]，其中GAI和RGA的获得性突变体极度矮化；水稻DELLA基因缺失突变体slr1(slender1)表现出细长的表型^[12]；玉米d8，d9突变体极度矮化^[1]；小麦RHT1基因获得一系列突变体(如rht-B1d，rht-B1b，rht-D1b，rht-D1d，rht-B1e和rht-B1c等)表现出高矮不一的表型^[13]. 除了对株高的调控外，DELLA基因还参与植物多方面的生长发育调控. 葡萄L1基因编码的DELLA序列突变为DELHA，使葡萄蔓藤上的蔓须变成了花序^[14]；在拟南芥中的研究也认为RGA，RGL1和RGL2基因参与花器官发育调控^[15]，转录因子WRKY75和GAI，RGA蛋白互作调控植株开花^[16]；RGL1和RGL2参与调控种子萌发^[17]. 这些研究报道表明DELLA基因与株高等多种重要性状相关.

最近，在大豆中报道了4个DELLA基因，即GmRGAa/b和GmRGLa/b. 其中，GmRGAs能够正调控转录因子GmSTF1/2的蛋白水平，而GmSTF1/2通过激活赤霉素代谢基因GmGA2ox-7b的表达使大豆株高降低^[18]. 为了进一步了解大豆DELLA基因的遗传变异，本文利用29个大豆种质资源的基因组数据，用生物信息学的方法鉴定了232个大豆DELLA基因家族成员，并分析了其生物学功能.

1. 材料与方法

1.1. 大豆种质资源基因组数据

在大豆SoyOmics^[19]数据库下载28个大豆种质资源的基因组数据，资源名称分别是Xu Dou No.1，Tie Feng No.18，Ju Xuan No.23，Wan Dou No.28，Amsoy，Yu Dou No.22，Jin Dou No.23，Qi Huang No.34，Han Dou No.5，PI 548362，Ji Dou No.17，Dong Nong No.50，Hei He No.43，Ke Shan No.1，Zhutwinning2，Zi Hua No.4，Tong Shan Tian E Dan，58-161，PI 398296，Zhang Chun Man Cang Jin，Feng Di Huang，Tie Jia Si Li Huang，Shi Sheng Chang Ye，PI 562565，PI 549046，PI 578357，W05，Zhonghuang 13，对应编号为SoyC01，SoyC02，SoyC03，SoyC04，SoyC05，SoyC06，SoyC07，SoyC08，SoyC09，SoyC10，SoyC11，SoyC12，SoyC13，SoyC14，SoyL01，SoyL02，SoyL03，SoyL04，SoyL05，SoyL06，SoyL07，SoyL08，SoyL09，SoyW01，SoyW02，SoyW03，W05，ZH13. 其中，SoyC表示栽培种，SoyL表示地方种，SoyW01，SoyW02，SoyW03，W05为野生大豆(Glycine soja Sieb. and Zucc.). 在Phytozome数据库中下载大豆Williams82 (Wm82)的基因组数据.

1.2. 大豆DELLA基因的鉴定

拟南芥DELLA蛋白GAI，RGA，RGL1，RGL2和RGL3的蛋白序列从拟南芥TAIR数据库中下载. 分别用这5个DELLA蛋白序列在Phytozome数据库中检索Wm82的基因组，筛选比对长度接近蛋白全长，编码蛋白序列一致性在45%以上的基因. 候选的大豆DELLA基因编码的蛋白序列在SMART蛋白数据库中进一步分析，鉴定是否属于DELLA蛋白家族成员.

1.3. 大豆DELLA基因的泛基因组分析

在大豆SoyOmics数据库中，用Wm82的DELLA基因分别检索28个种质资源的基因组数据，筛选覆盖度在85%以上，E值为0的基因，作为各大豆种质资源的DELLA基因. 用MegAlign软件分别对所有DELLA基因的DNA和蛋白质序列进行多序列比对和进化树分析.

1.4. 大豆DELLA蛋白质结构分析

用MegAlign软件对Wm82的DELLA蛋白序列进行比对，参照文献报道^[20]对DELLA蛋白的保守结构域进行比较.

1.5. 大豆DELLA基因表达模式分析

从SoyOmics数据库中查询Zhonghuang 13(ZH13)的转录组数据，根据FPKM值在图云平台(http://cloudtutu.com.cn/)绘制基因表达热图.

1.6. 蛋白互作网络分析

用Wm82的DELLA蛋白序列在STRING^[21]数据库中搜索存在互作关系的大豆蛋白，最低交互得分设定为0.400.

3. 讨论与结论

我国大豆单产偏低，增加种植密度提升单产，是当前提高大豆产量的一个重要途径. DELLA基因是赤霉素信号的负调控因子，参与了调控植物的株高和避荫性反应. 在绿色革命中，小麦RHT1基因不同类型的突变体出现一系列高矮不一的表型^{[1, 13]}，利用这些种质资源选育耐密植品种，增加种植密度，大幅提升了小麦产量. 因此，对大豆DELLA基因开展深入研究，创制耐密植的大豆种质资源，具有重要的意义.

本研究利用29个大豆种质资源的基因组数据，分析比较大豆种质资源间DELLA基因的亲缘关系和遗传变异情况，发现4个野生大豆在4号染色体上的GmRGLs基因编码的蛋白序列一致性较高，暗示这个基因在功能上可能比较保守. 而野生大豆中其他的DELLA基因和地方种、栽培种的相比遗传距离相对较远，表明这些基因在驯化过程中受到了选择. DELLA基因的这些遗传变异是否与野生大豆从藤状生长形态转变为直立生长形态有关，还需要深入研究. 同时，部分种质资源的DELLA基因发生了无义突变和翻译提前终止的移码突变，这些突变是否与种质资源株高、结实性等性状的变异相关，也需要进一步探索.

赤霉素调控了植物株高、开花、结实、根伸长等生长发育过程，而DELLA蛋白是赤霉素信号传递途径中的核心因子，大豆DELLA基因的表达模式与赤霉素调控生长发育的作用相符合. 对大豆DELLA基因的表达情况分析表明，位于11号染色体上的GmGAI1基因在所有植物组织中均高表达，可能发挥了主要作用，而11号染色体上的GmGAI1基因表达量稍弱，可能发挥次要作用，这与最近报道的GmRGAb对株高的调控作用要强于GmRGAa的结论一致^[18]. GmRGL1 (ZH13.Chr10)和GmRGL2 (ZH13.Chr06) 分别在发育中的籽粒和果荚中优势表达，这两个基因在籽粒和果荚发育中的功能还有待深入鉴定. 同时，通过蛋白互作分析，发现大豆DELLA蛋白能够与ABHD3和NF-Y等蛋白家族互作，这里面部分蛋白已被证明与花器官发育、逆境应答等途径相关，筛选的这些互作蛋白有利于进一步揭示DELLA蛋白的作用机理.

综合而言，本研究利用29个大豆种质资源的基因组数据信息，对大豆DELLA基因进行泛基因组分析，初步明确DELLA基因的遗传变异和进化关系；利用转录组数据对大豆DELLA基因在不同植物组织器官的表达情况进行了分析比较，推测大豆DELLA基因在植物生长发育的各阶段发挥不同的生物学功能；通过蛋白互作分析，为揭示DELLA蛋白的作用机制提供了新的候选因子. 这为明确大豆DELLA蛋白的生物学功能以及创制耐密植大豆种质提供了参考.

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Pan-genomic Comparison and Functional Analysis of DELLA Genes in Soybean