Cloning and Expression of VcILR1 in Blueberry
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摘要:
目的 克隆蓝莓（Vaccinium spp）生长素酰胺水解酶基因VcILR1，分析该基因在蓝莓花、叶、茎、果和根中的表达模式和经过赤霉素处理的青果和成熟果两个时期的表达情况，为进一步探究该基因功能和蓝莓胚珠败育的机制提供依据。 方法 以蓝莓果实的cDNA为模板克隆得到VcILR1基因，利用ProtParam等工具对VcILR1进行生物信息学分析，利用qRT-PCR方法对VcILR1基因在蓝莓不同组织及赤霉素处理的青果和成熟果中的表达量进行分析。 结果 成功克隆到蓝莓VcILR1基因，该基因含976 bp的开放阅读框（ORF），编码325个氨基酸，有两个保守结构域，分别是Peptidase_M20和M20_dimer；编码蛋白的分子质量35364.20 kDa，理论等电点5.67，不稳定系数43.57，脂肪系数88.55，亲水性平均值−0.054，属于不稳定亲水性蛋白。系统进化树分析表明蓝莓VcILR1基因与山茶科植物的茶树（Camellia sinensis）生长素酰胺水解酶（IAA-Leucine Resistant 1-like，ILL）基因亲缘关系较近。qRT-PCR分析结果显示，VcILR1在蓝莓花、叶、茎、果和根中均有表达，在叶片中表达量最高，具有组织特异性；赤霉素处理能上调果实中VcILR1的表达。 结论 赤霉素处理后蓝莓青果和成熟果中VcILR1的表达量均显著增高，推测赤霉素处理能够促进蓝莓果实中生长素含量的提高，从而抑制胚珠发育，导致果实无籽。 Abstract:Objective VcILR1 of blueberry was cloned to analyze the expressions in various plant organs as well as after being treated by gibberellin to decipher its function and association with ovule abortion. Method Using the cDNA of blueberry as the template, VcILR1 was cloned by RT-PCR. Bioinformatics on the amino acid sequence was performed utilizing ProtParam and other relevant tools. Expressions of the gene in the flowers, leaves, stems, fruits, and roots of the plant as well as those in green and ripe fruits after being treated by gibberellin were determined. Result VcILR1 was successfully cloned and found to contain 976 bp ORF encoding 325 amino acids with two conserved structural domains, peptidase_M20 and M20_dimer, a molecular mass of 35364.20 kDa, a theoretical isoelectric point of 5.67, an instability coefficient of 43.57, and a lipid coefficient of 88.55. It was an unstable protein with a hydrophilic mean value of −0.054. A phylogenetic tree analysis showed the gene to closely relate to Camellia sinensis IAA-Leucine Resistant 1-like (ILL) gene. The qRT-PCR analysis revealed that the gene was expressed tissue-specific with the highest in the leaves, and that the gibberellin treatment up-regulated the expression in the fruits. Conclusion The expressions of VcILR1 in the blueberries significantly increased after being treated by gibberellin. It suggested that the plant hormone could stimulate IAA generation in the fruits and might also affect the ovule development. -
Key words:
- Blueberry /
- VcILR1 gene /
- gene cloning /
- seedless
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图 1 VcILR1基因PCR扩增结果
M：Marker DL2000；1–2：无效条带；3–4：目的条带ILR1。
Figure 1. PCR amplification of VcILR1
M: Marker DL2000; 1-2: Invalid strips; 3-4: Target strip ILR1.
图 3 VcILR1蛋白磷酸化位点预测（A）和蛋白亲水/疏水性预测（B）
Figure 3. Predicted phosphorylation sites of VcILR1 protein (A) and protein hydrophilicity/hydrophobicity (B)
图 4 VcILR1蛋白的二级结构预测（A）和三级结构预测（B）
Figure 4. Predicted secondary structure (A) and tertiary structure (B) of VcILR1 protein
图 5 蓝莓VcILR1基因与其他物种ILL家族基因的系统发育树
Figure 5. Phylogenetic trees of VcILR1 and ILL familial genes of other species
图 6 蓝莓VcILR1氨基酸序列与其他物种ILL家族蛋白氨基酸序列对比分析
以相似性 50%为阈值，蓝色标注：相似性 ≥ 50%；粉色标注：相似性 ≥ 75%；黑色标注：相似性 ≥100%；红线、蓝线：保守结构域。
Figure 6. Amino acid sequence alignment of VcILR1 with other ILL family proteins
With threshold at 50 % similarity, blue indicates≥50%; pink, ≥75%; black, 100%; red and blue lines, conservative domains.
图 7 VcILR1在蓝莓不同部位（A）和赤霉素处理下的相对表达量（B）
A：不同的小写字母表示不同部位间差异显著（P＜0.05）。B：青果CK——对照组青果期果实；青果GA——赤霉素处理组青果期果实；熟果CK——对照组成熟果实；熟果GA——赤霉素处理组成熟果实。不同小写字母表示同一阶段果实不同处理间差异显著（P＜0.05）。
Figure 7. Expressions of VcILR1 in different organs of blueberry plant (A) and of that being treated by gibberellin (B)
A: Data with different lowercase letters indicate significant difference between organs (P＜0.05); B: Green fruit CK—control green fruit stage; green fruit GA—gibberellin treated group green fruit stage; ripe fruit CK—control ripe fruit; ripe fruit GA—gibberellin treated group ripe fruit. Data with different lowercase letters indicate significant difference between treatments on fruits at the same growth stage (P＜0.05).
表 1 VcILR1基因扩增引物信息
Table 1. VcILR1 primer information
引物名称Primer name 引物序列（5′-3′）primer sequence（5′-3′） VcILR1-F ATGGATGCTTTGCCCATTCAGGA VcILR1-R AGAATAAGAAATGGGGTTTCTTCC qRT-VcILR1-F GAGGAACAAGGCCAAGGTGCAA qRT-VcILR1-R CCTGGCCTTGCTGCAACAGT Actin-F AGGCTAACCGTGAGAAGATGAC Actin-R AGAGTCCAGCACGATTCCAG 
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