滇山茶bHLH基因家族鉴定及花色形成相关基因筛选

doi:10.13560/j.cnki.biotech.bull.1985.2024-0257

摘要/Abstract

摘要：

【目的】通过生物信息学方法初步筛选与滇山茶花色相关的bHLH基因，为后续深入研究滇山茶花色提供支持。【方法】基于滇山茶全长转录组数据，通过生物信息学方法鉴定滇山茶bHLH基因家族成员，并对其理化性质、二级结构、系统发育关系和结构域等进行分析。利用二代转录组和代谢组数据挖掘与滇山茶花色相关的bHLH基因，通过实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）验证bHLH基因在滇山茶开花过程中和不同品种间的表达情况。【结果】在滇山茶全长转录组中鉴定到71个滇山茶CrbHLHs基因，蛋白分子量的范围在22 994.58-102 996.96 Da，等电位为4.98-9.51，均为亲水性蛋白。与拟南芥聚类分析发现，滇山茶bHLH基因家族成员可分布到14个亚族。多组学联合分析发现，CrbHLH8、CrbHLH61和CrbHLH63与矢车菊色素苷呈正相关，而CrbHLH59与其呈负相关；CrbHLH13和CrbHLH71与原花青素呈正相关，其中CrbHLH8和CrbHLH61为IIIf亚族成员。【结论】从滇山茶全长转录组中鉴定出71个CrbHLHs家族成员，其中6个CrbHLHs与滇山茶花色相关。

关键词: 滇山茶, bHLH基因家族, 花色, 进化树, 全长转录组, 生物信息学

Abstract:

【Objective】The preliminary screening of bHLH genes related to C. reticulata flower color by bioinformatics method provides support for further study of C. reticulata flower color.【Method】Based on the full-length transcriptome data of C. reticulata, the members of the bHLH gene family were identified by bioinformatics method, and their physical and chemical properties, secondary structure, phylogenetic relationships and domains were analyzed. The second-generation transcriptome and metabolome data were used to mine the bHLH genes related to the flower color of C. reticulata. Real-time quantitative polymerase chain reaction（RT-qPCR）was used to verify the expression of bHLH gene during flowering of C. reticulata among different varieties.【Result】Total 71 CrbHLHs genes were identified in the full-length transcriptome of C. reticulata. Their molecular weight ranged from 22 994.58 to 102 996.96 Da, and their equipotential ranged from 4.98 to 9.51. All of them were hydrophilic proteins. The cluster analysis with Arabidopsis thaliana showed that the bHLH gene family members of C. reticulata could be distributed into 14 subfamilies. Multiple omics analysis showed that CrbHLH8, CrbHLH61 and CrbHLH63 were positively correlated with cyanidin, while CrbHLH59 was negatively correlated with cyanidin. CrbHLH13 and CrbHLH71 were positively correlated with proanthocyanidins, and CrbHLH8 and CrbHLH61 were members of the IIIf subfamily.【Conclusion】The 71 CrbHLHs family members were identified from the full-length transcriptome of C. reticulata, of which 6 CrbHLHs were related to C. reticulata flower color.

Key words: Camellia reticulata, bHLH gene family, flower color, phylogenetic tree, full-length transcriptome, bioinformatics

周麟, 黄顺满, 苏文坤, 姚响, 屈燕. 滇山茶bHLH基因家族鉴定及花色形成相关基因筛选[J]. 生物技术通报, 2024, 40(8): 142-151.

ZHOU Lin, HUANG Shun-man, SU Wen-kun, YAO Xiang, QU Yan. Identification of the bHLH Gene Family and Selection of Genes Related to Color Formation in Camellia reticulata[J]. Biotechnology Bulletin, 2024, 40(8): 142-151.

图/表 9

表1 RT-qPCR引物

Table 1 Primers used for RT-qPCR

基因Gene	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
CrbHLH8	GCAGTTGTGCCCTCAACAAG	CACTCACCTCCATTGGCTGT
CrbHLH13	CGGCTGAATGCTCGTTGATG	CTGAAATTGGCGGTGTTGGG
CrbHLH59	ACCCCATTGAATTCCCCACC	TACAAATCCAGGATCCGCGG
CrbHLH61	GCAGTTGTGCCCTCAACAAG	CACTCACCTCCATTGGCTGT
CrbHLH63	TGTCCATTGGATGCTCACCC	CAGCACCATCTTGAGTCCGT
CrbHLH71	AGCCACGTACTGCTGAACAA	TCTGCGCTATTGCCAGAACA
CrActin	GTCTGTTCCCTCTCCTCCCT	GAAGGGATCGTTGACAGCGA

图1 滇山茶与拟南芥bHLH基因家族系统进化树

Fig. 1 Phylogenetic tree of bHLH gene family of C. reticulata and A. thaliana

图2 滇山茶和其他物种bHLH家族IIIf亚族成员系统进化树

Fig. 2 Phylogenetic tree of IIIf subgroup members of bHLH family in C. reticulata and other species

图3 滇山茶bHLH基因家族的保守基序和保守结构域分析 A：滇山茶bHLH基因家族成员聚类分析；B：滇山茶bHLH基因成员的保守基序分析；C：滇山茶bHLH基因家族成员的保守结构域

Fig. 3 Analysis of conserved motif and conserved domain of the bHLH gene family of C. reticulate A: Cluster analysis of bHLH gene family members of C. reticulata. B: Conserved motif analysis of bHLH gene members of C. reticulata. C: Conserved domain of the bHLH gene family members of C. reticulata

图4 滇山茶差异bHLH分析 A：滇山茶品种间Veen图；B：滇山茶不同发育时期Veen图；C：差异bHLH基因组合热图；S：狮子头；T：童子面；1：花蕾期；2：盛花期；-1、-2、-3表示3个重复。下同

Fig. 4 Differential bHLH analysis of C. reticulate A: Veen map of C. reticulata varieties. B: Veen map of C. reticulata at different developmental stages. C: Differential bHLH gene combination heat map. S: C. reticulata ‘Shizitou’. T: C. reticulata ‘Tongzimian’. 1: Bud stage. 2: Full flowering stage. -1, -2, and -3 indicate three repetitions. The same below

图5 滇山茶‘狮子头’和‘童子面’花色表型（A）及关键色素含量（B）不同小写字母表示在P<0.05水平差异显著。下同

Fig. 5 Floral phenotype（A）, and key pigment content（B）in C. reticulata ‘Shizitou’ and C. reticulata ‘Tongzimian’ Different lower letters indicate significant differences at P<0.05 level. The same below

图6 滇山茶bHLH与矢车菊色素苷和原花青素的网络调控图

Fig. 6 Network regulation of C. reticulata's bHLH with cyanidin glycosides and proanthocyanidins

图7 滇山茶bHLH家族基因表达模式分析

Fig. 7 Analysis of gene expression pattern in the bHLH family of C. reticulata

图8 滇山茶bHLH调控花色通路合成通路

Fig. 8 bHLH regulates the synthesis pathway of color pathways in C. reticulata

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