Whole-genome analysis of the <i>HubHLH</i> gene family in Pitaya and its differential expression in response to supplementary light-induced flowering in winter

LI Jiaxue; DING Yi; WANG Meng; LI Tao; GUO Panyang; LIU Chengli; WEI Shuangshuang; HUANG Jiaquan; LI Hongli; HU Wenbin; TANG Hua

doi:10.15886/j.cnki.rdswxb.20220108

Volume 15 Issue 2

Mar. 2024

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Article Navigation > Journal of Tropical Biology > 2024 > 15(2): 198-209

LI Jiaxue, DING Yi, WANG Meng, LI Tao, GUO Panyang, LIU Chengli, WEI Shuangshuang, HUANG Jiaquan, LI Hongli, HU Wenbin, TANG Hua. Whole-genome analysis of the HubHLH gene family in Pitaya and its differential expression in response to supplementary light-induced flowering in winter[J]. Journal of Tropical Biology, 2024, 15(2): 198-209. doi: 10.15886/j.cnki.rdswxb.20220108

Citation:

LI Jiaxue, DING Yi, WANG Meng, LI Tao, GUO Panyang, LIU Chengli, WEI Shuangshuang, HUANG Jiaquan, LI Hongli, HU Wenbin, TANG Hua. Whole-genome analysis of the HubHLH gene family in Pitaya and its differential expression in response to supplementary light-induced flowering in winter[J]. Journal of Tropical Biology, 2024, 15(2): 198-209. doi: 10.15886/j.cnki.rdswxb.20220108

Whole-genome analysis of the HubHLH gene family in Pitaya and its differential expression in response to supplementary light-induced flowering in winter

doi: 10.15886/j.cnki.rdswxb.20220108

LI Jiaxue^{1, 2
,},
DING Yi^{1, 2},
WANG Meng^{1, 2},
LI Tao^{1, 2},
GUO Panyang²,
LIU Chengli²,
WEI Shuangshuang²,
HUANG Jiaquan^{1, 2},
LI Hongli³,
HU Wenbin³,
TANG Hua^{1, 2
,
,}

1.
Sanya Nanfan Research Institute of Hainan University/ Hainan Yazhou Bay Seed Laboratory, Sanya, Hainan 572025, China
2.
College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China
3.
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571700, China

Received Date: 2022-12-06
Rev Recd Date: 2023-05-04

Available Online: 2023-12-14

Publish Date: 2024-03-25

Abstract

In order to obtain relative complete candidate genes, the HubHLH gene expression response in Pitaya during the flowering process induced by supplementary light in winter was investigated, and the whole genome of HubHLH gene family in Pitaya was analyzed. There were 153 pitaya HubHLH genes identified, and their encoding proteins contained 176-687 amino acids with their molecular weight being 19.28 – 74.44 kDa, pI 4.81-9.88. These proteins were all hydrophilic. The prediction of subcellular localization showed that most of the proteins were localized in the nucleus. The pitaya HubHLH proteins were compared with 120 Arabidopsis AtbHLH proteins for phylogenetic analysis. Phylogenetic analysis showed that the pitaya HubHLH proteins were divided into 12 groups and 25 subfamilies. Analysis of conserved motif, gene structure and location distribution in chromosomes showed that genes of the same subfamily had similar motif composition and gene structure. Analysis of the internal replication events of the pitaya HubHLH gene family found that 78 genes were identified as fragment repeating genes, indicating that fragment replication was the main expansion force of the pitaya HubHLH gene family. In addition, based on the transcriptomic data of the four phases of supplementary light-induced flowering of Pitaya in winter, 59 HubHLH genes were differentially expressed during the process of supplementary light induced flowering in winter. GO function enrichment showed that they were enriched in response to red or far-red light, light stimulation, sexual reproduction function, and radiation. The HubHLH gene family may play a regulatory role in the flowering process of Pitaya induced by supplementary light in winter. In this study, we conducted a comprehensive genome analysis of the HubHLH gene family in Pitaya, obtained relatively complete candidate genes, and preliminarily investigated the expression response of HubHLH genes in the winter flowering process induced by supplementary light in Pitaya.
- pitaya,
- HubHLH gene family,
- whole genome analysis,
- supplementary light-induced flowering,
- differential gene expression

References

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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火龙果（ Hylocereus undatus ）原产中美洲热带地区，是仙人掌科（Cactaceae）量天尺属（Hylocereus）或蛇鞭柱属（Selenicereus）植物，喜光喜热。火龙果在我国的种植面积已达到6.7万公顷，在热区经济中已占有重要地位^[1]。火龙果是长日照作物，在我国因冬季光照时长不足，不能开花，为此在生产上可采用夜晚补光的方式诱导火龙果开花结果。bHLH转录因子蛋白，包含1个碱性DNA结合区和1个螺旋－环－螺旋（HLH）区，能通过与顺式启动子元件相结合来调节基因表达，参与植物开花、种子发芽、矿质营养与非生物胁迫、应激反应、光信号、激素信号、光和植物激素之间的相互作用等多种生理过程^[2]。

bHLH基因家族参与调控植物的开花调控，在拟南芥、番茄等植物中已有广泛的研究。在成花起始阶段，拟南芥的FBH蛋白（bHLH类型转录因子），可与光周期信号途径的关键基因CO （CONSTANS）启动子的E-box顺式元件结合，激活光周期开花基因的共转录^[3]。在花发育阶段，与CIB1同源的bHLH蛋白，可以作为激活子直接促进成花素基因FT的转录^[4]。bHLH家族的光敏色素互作因子PIFs可以与光信号直接作用，PIF3在光感受器信号网络中直接与光敏色素反应，PIF4在高温下直接激活成花素基因FT^[5]。bHLH48和bHLH60与PIF7联合，可以作为赤霉素途径中的正调控因子，正调控GA介导的开花^[6]。bHLH38/100/101会干扰CO的转录，调节FT的表达，进而影响拟南芥的开花^[7]。bHLH基因家族成员SlbHLH22的过表达，会促进番茄早开花^[8]。在茉莉酸途径中，JA激活的bHLH转录因子MYC2, MYC3和MYC4冗余调控拟南芥的开花^[9]。拟南芥的bHLH家族基因BEE1具有诱导开花起始的功能，BES1-BEE1-FT可以调节光周期并与FT的激活因子CO互作来影响植物开花^[10]。bHLH基因家族CRY2、CIB1可以响应蓝光，能将生物钟信号转化为开花信号，促进CO基因形成蛋白质复合物，诱导花的形成^[11]。除了调节开花时间外，bHLH基因家族成员的SPT基因可以在花器官形成中起作用^[12]。

目前，bHLH基因家族在拟南芥（Arabidopsis thaliana）^[13]、小麦（Triticum aestivum L.）^[14]、谷子（Setaria italica L.）^[15]、烟草（Nicotiana tabacum L.）^[16]、柚子（Citrus maxima）^[17]等植物中进行了全基因组鉴定。火龙果作为一种重要的热带水果，有独特的开花习性，bHLH家族基因可能发挥重要作用，但目前还未见火龙果HubHLH基因家族相关系统分析的研究报道。火龙果（Hylocereus undatus）全基因组序列的发表，为鉴定火龙果HubHLH基因家族提供了条件^[18]。本研究拟利用火龙果（Hylocereus undatus）基因组数据，对火龙果HubHLH转录因子家族进行全基因组筛选，并进行理化性质、系统进化、基因表达等方面的比较分析；利用火龙果补光诱导开花的转录组数据，研究HubHLH基因家族在火龙果冬季补光诱导开花过程的表达响应，旨在解析火龙果冬季补光诱导开花的分子机制。

3. 讨　论

3.1. 火龙果与拟南芥HubHLH基因家族的系统发育关系

bHLH作为植物中第二大类转录因子，近几年在菠萝（Ananas comosus L.）^[25]、香蕉（Musa acuminata L.）^[26]、木薯（Manihot esculenta crantz）^[27]等热带植物中实现了全基因组鉴定，分别鉴定出121、259、148个bHLH基因。火龙果全基因组序列的发表，为进一步检索、克隆和验证火龙果的bHLH基因提供了条件。同时，冬季补光诱导开花过程是严密而复杂的过程，bHLH基因被证实参与了植物生长发育的很多过程。因此对火龙果进行bHLH基因家族的全基因组筛选和分析，为后续它们对冬季补光诱导开花的响应过程中的功能研究很有必要。本研究首次对火龙果bHLH基因家族进行了全基因组鉴定，得到153个火龙果HubHLH基因，对它们的理化性质全面预测，得到火龙果HubHLH的分子量、氨基酸长度、等电点等理化性质，与其他植物中的bHLH差异不大，亚细胞定位预测主要分布在细胞核。

目前，植物中bHLH基因还未有特别明确的分类标准，通常选择与模式植物或亲缘关系相近的植物，共同构建有根系统发育树进行分类，本研究选择长日照植物的拟南芥，将它的120个bHLH蛋白序列和鉴定到的153个火龙果HubHLH蛋白序列共同构建有根系统发育树，通过与拟南芥的系统发育关系比较，火龙果bHLH分类结果和拟南芥几乎一致，都得到了12个主要的组别（Ⅰ-Ⅻ）^[28]。进行火龙果HubHLH基因家族的保守motif和基因结构分析，153个HubHLH基因都含有motif 1和motif 2，同一分组的基因结构相似，证明了系统发育分析结果的可靠性。motif位点的识别有助于鉴定其家族同源性和预测蛋白质的功能，而系统发育树显示，一些火龙果中的HubHLH与拟南芥的bHLH基因可能同源，并具有相似功能，因此可以通过发现聚集到同一亚家族的拟南芥中功能较为明确的bHLH蛋白，发现火龙果中bHLH的可能功能。例如在亚家族IIIe中的成员与茉莉酸介导的开花相关，亚家族Ⅶ的成员多与光信号有关^[29]，成员较多的Ⅻ亚家族中有些调节光周期开花^[30]，有些与光和植物激素之间的相互作用有关^[31]。

3.2. 火龙果HubHLH基因的差异表达及GO、KEGG富集分析

基于转录组数据展示HubHLH基因家族成员在火龙果冬季夜晚补光诱导成花过程中NL、L0、L1、L2四个时期的差异表达情况，其中有注释到JA途径中调节开花时间的MYC2基因，在补光后表达含量急速增加；还有响应光和温度，在花形态发生过程中起作用的光稳定SPATULA基因，也在L0-L1阶段表达出上调趋势，其他HubHLH基因对补光诱导也有不同程度的响应。另外，根据HubHLH差异基因的GO富集结果，发现在有性生殖，即火龙果成花过程，对光反应和光刺激，即补光过程，都得到了功能富集。HubHLH基因家族可能在冬季补光诱导火龙果成花过程起到了作用。总之，开花时间的调控是严格而复杂的，在冬季通过补光诱导火龙果开花具有重要的生产实践价值，本研究利用火龙果基因组信息和转录组信息，对火龙果HubHLH基因家族进行了全面分析研究，为火龙果冬季补光诱导开花，提供了许多潜在的候选基因。

Reference (31)

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[2]	HAO Y, ZONG X, REN P, et al. Basic helix-loop-helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis[J]. Int J Mol Sci, 2021, 22(13): 7152.
[3]	ITO S, SONG Y H, JOSEPHSON-DAY A R, et al. FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis[J]. Proc Natl Acad Sci U S A, 2012, 109(9): 3582 − 3587.
[4]	LIU H, YU X, LI K, et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis[J]. Science, 2008, 322(5907): 1535 − 1539.
[5]	KUMAR S V, LUCYSHYN D, JAEGER K E, et al. Transcription factor PIF4 controls the thermosensory activation of flowering[J]. Nature, 2012, 484(7393): 242 − 245.
[6]	LI Y, WANG H, LI X, et al. Two DELLA-interacting proteins bHLH48 and bHLH60 regulate flowering under long-day conditions in Arabidopsis thaliana[J]. J Exp Bot, 2017, 68(11): 2757 − 2767.
[7]	CHEN W, ZHAO L, LIU L, et al. Iron deficiency-induced transcription factors bHLH38/100/101 negatively modulate flowering time in Arabidopsis thaliana[J]. Plant Sci, 2021, 308: 110929.
[8]	WASEEM M, LI N, SU D, et al. Overexpression of a basic helix-loop-helix transcription factor gene, SlbHLH22, promotes early flowering and accelerates fruit ripening in tomato (Solanum lycopersicum L.)[J]. Planta, 2019, 250(1): 173 − 185.
[9]	WANG H, LI Y, PAN J, et al. The bHLH Transcription Factors MYC2, MYC3, and MYC4 are required for jasmonate-mediated inhibition of flowering in Arabidopsis[J]. Mol Plant, 2017, 10(11): 1461 − 1464.
[10]	WANG F, GAO Y, LIU Y, et al. BES1-regulated BEE1 controls photoperiodic flowering downstream of blue light signaling pathway in Arabidopsis[J]. New Phytol, 2019, 223(3): 1407 − 1419.
[11]	LIU Y, LI X, MA D, et al. CIB1 and CO interact to mediate CRY2-dependent regulation of flowering[J]. EMBO Rep, 2018, 19(10): e45762.
[12]	REYMOND M C, BRUNOUD G, CHAUVET A, et al. A light-regulated genetic module was recruited to carpel development in Arabidopsis following a structural change to SPATULA[J]. Plant Cell, 2012, 24(7): 2812 − 2825.
[13]	HEIM M A, JAKOBY M, WERBER M, et al. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[J]. Mol Biol Evol, 2003, 20(5): 735 − 747.
[14]	GUO X J, WANG J R. Global identification, structural analysis and expression characterization of bHLH transcription factors in wheat[J]. BMC Plant Biol, 2017, 17(1): 90.
[15]	FAN Y, LAI D, YANG H, et al. Genome-wide identification and expression analysis of the bHLH transcription factor family and its response to abiotic stress in foxtail millet (Setaria italica L.)[J]. BMC Genomics, 2021, 22(1): 778.
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