甜菜BvATGs基因家族全基因组鉴定及盐胁迫下表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0504

摘要/Abstract

摘要：

目的自噬是维持生物体内细胞稳态的重要降解途径，在调控植物响应逆境胁迫中起着重要作用。挖掘和鉴定甜菜（Beta vulgaris L.）自噬相关基因（autophagy-related genes, ATGs）家族成员，并分析其在盐胁迫下的表达模式，为探究BvATGs在逆境下的功能提供理论依据。方法利用生物信息学方法从甜菜基因组中鉴定BvATGs基因家族成员，分析其蛋白质理化性质、染色体分布、系统发育、基因结构、保守基序、顺式调控元件和共线性；采用RT-qPCR分析BvATGs在盐胁迫下的表达模式；采用PEG介导法瞬时转化拟南芥原生质体，以确定BvATG4和BvATG6a-1亚细胞定位；构建BvATG4和BvATG6a-1过表达载体并遗传转化拟南芥。结果鉴定出51个BvATGs基因，分为20个亚家族；其中，48个BvATGs不均地分布在9条染色体上，而3个基因（BvATG1a、BvATG1d和BvATG1k）未定位在染色体；同一亚家族内的BvATGs基因具有相似的基因结构和保守域。BvATGs编码蛋白质氨基酸数为84‒2 467 aa，分子质量为10.21‒277.30 kD，大部分为亲水性蛋白质；88.2%的BvATGs成员定位于细胞质、细胞核和叶绿体。通过甜菜物种内共线性分析发现BvATGs有3对同源基因，物种间共线性显示BvATGs在水稻和拟南芥中分别存在9对和30对同源基因；在BvATGs启动子区含有大量的光响应元件、激素响应元件和逆境胁迫响应元件。RT-qPCR分析显示，盐胁迫处理下，12个BvATG基因在叶和根中的表达量均有不同程度上调。BvATG4主要定位于细胞核和细胞质，BvATG6a-1主要定位于细胞质及内质网。与野生型拟南芥相比，转基因株系中BvATG4和BvATG6a-1基因相对表达水平均显著增加。结论从甜菜全基因组中鉴定出51个BvATGs基因家族成员，其中12个基因不同程度地受盐胁迫诱导和上调。BvATG4定位在细胞核和细胞质，而BvATG6a-1定位在细胞质和内质网。进一步将BvATG4和BvATG6a-1转入拟南芥，分别获得高表达的转基因株系OE4和OE2。为甜菜耐盐基因资源挖掘与利用奠定基础。

关键词: 甜菜, 生物信息学, 自噬, 自噬相关基因, 盐胁迫, 表达分析, 亚细胞定位

Abstract:

Objective Autophagy is a crucial degradation pathway for maintaining cellular homeostasis and plays an essential role in plant responses to abiotic stress. In this study, autophagy-related genes (ATGs) were identified and characterized in sugar beet (Beta vulgaris L.), and their expression patterns under salt stress were analyzed to provide a theoretical basis for understanding their potential functions in stress responses. Method Members of the BvATGs gene family were identified from the sugar beet genome using bioinformatics methods, and the physicochemical properties, chromosomal distribution, phylogenetic relationships, gene structures, conserved motifs, cis-acting regulatory elements, and collinearity were analyzed. RT-qPCR was used to analyze the expression patterns of these genes under salt stress. The subcellular localization of BvATG4 and BvATG6a-1 was determined through transient transformation of Arabidopsisthaliana protoplasts mediated by PEG. Furthermore, the overexpression vectors for BvATG4 and BvATG6a-1 were constructed and genetically transformed into A.thaliana. Result A total of 51 BvATGs genes were identified and classified into 20 subfamilies in sugar beet. Among them, 48 BvATGs were unevenly distributed across nine chromosomes, while the other three genes (BvATG1a, BvATG1d and BvATG1k) were not mapped onto chromosomes. The BvATGs genes in the same subfamily had similar gene structures and conserved domains. The number of amino acids encoding BvATGs genes was 84-2 467 aa, and the molecular weight was 10.21-277.30 kD, most of which were hydrophilic proteins. Approximately 88.2% of the BvATGs members were localized to the cytoplasm, nucleus and chloroplast. Intraspecific collinearity analysis showed that there were 3 pairs of homologous genes of BvATGs, and interspecific collinearity showed that there were 9 pairs and 30 pairs of homologous genes of BvATGs in rice (Oryza sativa) and A. thaliana, respectively. A large number of light-responsive, hormone-responsive and stress-responsive elements were found in the promoter regions of the BvATGs genes. The RT-qPCR analysis showed that the expressions of 12 BvATG genes in the shoots and roots were up-regulated to varying degrees under salt stress. BvATG4 was primarily localized in the nucleus and cytoplasm, while BvATG6a-1 was mainly localized in the cytoplasm and endoplasmic reticulum. Compared to wild-type A. thaliana, the relative expressions of BvATG4 and BvATG6a-1 genes in the transgenic lines significantly increased. Conclusion A total of 51 BvATGs family members are identified at the whole genome of sugar beet in this study. Among those, 12 genes are differentially induced and up-regulated under salt stress. BvATG4 is localized in the nucleus and cytoplasm, while BvATG6a-1 is localized in the cytoplasm and endoplasmic reticulum. Further, BvATG4 and BvATG6a-1 are introduced into A. thaliana, and transgenic lines with high expression (OE4 and OE2) are obtained, respectively. This study establishs a theoretical basis for identification and utilization of salt-tolerant genetic resources in sugar beet.

Key words: sugar beet, bioinformatics, autophagy, autophagy-related genes, salt stress, expression analysis, subcellular localization

任云儿, 伍国强, 成斌, 魏明. 甜菜BvATGs基因家族全基因组鉴定及盐胁迫下表达模式分析[J]. 生物技术通报, 2026, 42(1): 184-197.

REN Yun-er, WU Guo-qiang, CHENG Bin, WEI Ming. Genome-wide Identification of the BvATGs Genes Family in Sugar Beet （Beta vulgaris L.） and Analysis of Their Expression Pattern under Salt Stress[J]. Biotechnology Bulletin, 2026, 42(1): 184-197.

图/表 10

表1 本研究所用RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR in this study

基因名称 Gene name	上游引物 Forward primer sequence （5′‒3′）	下游引物 Reverse primer sequence （5′‒3′）
BvACTIN	ACTGGTATTGTGCTTGACTC	ATGAGATAATCAGTGAGATC
BvATG1e	CAATCTGTCTGGCTTATTCGATG	CGCTCGAAGTTTAGCAACCC
BvATG1h	TTTGCCGGAACACCTTAGACA	CCTTAGCACCATCATATCCCT
BvATG3	CCATGCTCGCAAAACGGTA	GTCAACTTCTGGCTCAACCC
BvATG4	TCGTCGTCAACTAGGGCTTC	CTCCTCCGAACGATGCTCA
BvATG6a-1	GTCTCCCTAAAGTTCCGGTTG	TTTACAGGCCCAAACAGGT
BvATG8b	CCTACTGGAGCAATCATGTCT	CTCCACAGCAAGATACCCGAA
BvATG8c	GCTCCAACATCCTCTCGAAC	TGCCCCACAGTTAAATCAGC
BvATG8d	TCCTGAGAAAGCCATATTCGT	TAGCAATGCACCTCAATCCAA
BvATG8e	TGCCAAGTATCCTGATCGAGT	ATATAAATAGAGCTTTGCCCGGAG
BvATG10	CCTCTCTTCACTTCCCACGA	TTGCAGGATCAATACGCTCT
BvATG16-2	CAGTGCAGCTTATGATCGAAC	CAAAGCCGGAGATTCCCAT
BvATG20c	TCCCGAGATTTCCACTACTGC	AAGGATCGAAACATAGCGTCA

表2 本研究中所用的引物序列

Table 2 Primers used in the study

引物用途 Primer purpose	引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
基因克隆 Gene clone	BvATG4-F-Xho I	GAGGACACGCTCGAGATGAAGAACTTGTTTGAGAGTGCTGG
	BvATG4-R-Sma I	CTTTGTAATCCCCGGGGAGGAGTTGCCATTCGTCCTCTTG
	BvATG6a-1-F-Xho I	TTTGGAGAGGACACGCTCGAGATGAAGGAGAAGCAGCTTG
	BvATG6a-1-R-Sma I	TCATCTTTGTAATCCCCGGGTTGACCAGATGGAAATCGAG
转基因拟南芥PCR检测 Transgenic A. thaliana PCR detection	35S-F	GACGCACAATCCCACTATCC
	BvATG4-R	GAGGAGTTGCCATTCGTCCTCTTG
	BvATG6a-1-R	TTGACCAGATGGAAATCGAG
内参基因 Refenrence gene	AtUBQ10-F	AGATCCAGGACAAGGAAGGTATTC
内参基因 Refenrence gene	AtUBQ10-R	CGCAGGACCAAGTGAAGAGTAG
亚细胞定位 Subcellular localization	BvATG4-F-Hind III	GGACAGCCCAAGCTTATGAAGAACTTGTTTGAGAGTGCTGG
	BvATG4-R-Nco I	TTGCTCACCATGGATCCGAGGAGTTGCCATTCGTCCTCTTG
	BvATG6a-1-F-Hind III	CATGGAGGCCAGTGAATTCATGAAGGAGAAGCAGCTTGATG
	BvATG6a-1-R-Nco I	TCGAGCTCGATGGATCCTTGACCAGATGGAAATCGAGAATC

图1 BvATGs的染色体定位

Fig. 1 Chromosome location of BvATGs

图2 高等植物ATGs系统进化分析黄色五角星代表甜菜，绿色圆圈表示苜蓿，蓝色三角形表示谷子；ATGs基因名称和登录号见附表2

Fig. 2 Phylogenetic analysis of ATGs in higher plantsThe yellow pentagons indicate sugar beet (B. vulgaris), the green circles indicate M. truncatula and the blue triangles indicate S. italica. The name and accession number of ATGs are shown in the Supplementary Table S2

图3 BvATGs家族保守结构域（A）和基因结构（B）分析

Fig. 3 Analysis of conserved domains (A) and gene structure (B) of BvATGs family

图4 BvATGs基因启动子顺式作用元件预测分布（A）和数量（B）

Fig. 4 Predicting distribution (A) and number (B) of cis-acting elements in the promoters of BvATGs genes

图5 甜菜BvATGs基因家族的共线性分析A：ATGs基因家族种内共线性分析；B：甜菜与拟南芥和水稻之间ATG基因的共线性分析

Fig. 5 Collinearity analysis of BvATGs gene family in sugar beetA: ATGs gene family intraspecies collinearity analysis. B: Synteny analysis of ATG genes between sugar beet (B. vulgaris), rice (O. sativa) and A. thaliana

图6 NaCl处理12 h （A）和24 h （B）的甜菜叶片和根中BvATGs相对表达水平将数据标准化为BvACTIN表达水平。竖线表示标准误差（SE）（n = 3）。柱子上不同小写字母表示P < 0.05水平差异显著。下同

Fig. 6 Relative expressions of BvATGs in the shoots and roots of sugar beet plants treated with different concentrations of NaCl for 12 h (A) and 24 h (B)Data are normalized to BvACTIN expression. Vertical bars indicate standard error (SE) (n = 3). Different lowercase letters on the bars indicate significant difference at P < 0.05 level. The same below

图7 BvATG4 （A）和BvATG6a-1 （B）的亚细胞定位

Fig. 7 Subcellular localization of BvATG4 (A) and BvATG6a-1 (B)

图8 BvATG4和BvATG6a-1转基因拟南芥鉴定A：BvATG4和BvATG6a-1转基因拟南芥基因组DNA PCR检测；B：BvATG4和BvATG6a-1转基因拟南芥RT-qPCR检测；M：DL2000分子量标记物；+：pEG-BvATG4、pEG-BvATG6a-1质粒；WT：野生型拟南芥；1‒10：BvATG4和BvATG6a-1转基因拟南芥；OE4、OE7、OE9：BvATG4转基因拟南芥；OE2、OE3、OE4：BvATG6a-1转基因拟南芥。将数据标准化为AtUBQ1表达水平

Fig. 8 Identification of BvATG4 and BvATG6a-1 transgenic A. thalianaA: Genomic DNA PCR detection of BvATG4 and BvATG6a-1 transgenic A. thaliana. B: RT-qPCR detection of BvATG4 and BvATG6a-1 transgenic A. thaliana. M: DL2000 marker for molecular weight; + : pEG-BvATG4 and pEG-BvATG6a-1 plasmid. WT: Wild-type A. thaliana; 1-10: BvATG4 and BvATG6a-1 transgenic A. thaliana; OE4, OE7, OE9: BvATG4 transgenic A. thaliana; OE2, OE3, OE4: BvATG6a-1 transgenic A. thaliana

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