Identification of ChSEP genes in the whole genome of hazelnut, preparation of polyclonal antibody and expression in ovules
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摘要:
目的 正常的胚珠充实是榛子种仁形成的前提与基础，胚珠充实不足可频繁导致瘪仁、空壳果实的形成，SEP基因被认为是可以影响胚珠发育的E类MADS-box基因。为研究SEP在榛子种仁充实过程中的重要调节作用，拟鉴定参与榛子胚珠发育的重要作用基因ChSEP，并制备ChSEP的多克隆抗体，开展正常发育与败育胚珠中ChSEP的免疫组织化学分析，以期为深入解析SEP基因调控榛子胚珠发育机制提供科学依据。 方法 用榛子基因组与转录组数据鉴定基因组中所有的SEP基因家族成员，并进行重要SEP基因的原核表达载体构建、基因表达及多克隆抗体制备，最后对参与胚珠发育调控的重要SEP蛋白进行免疫组织化学检测。 结果 （1）榛子基因组中鉴定到3条ChSEP蛋白序列（Cor0054610.1、Cor0008190.1、Cor0119400.1），所有ChSEP蛋白均包含SRF-TF与K-box结构域。制备了Cor0054610.1的多克隆抗血清E18305和E18306，它们的抗体抗原条带分子量在45 kDa左右，1∶1 000稀释后可检测到1 ~ 2 ng抗原。（2）免疫组织化学分析结果表明：ChSEP在发育胚珠与败育胚珠的珠被中均有表达，珠被中的ChSEP表达量高于子叶，正常发育胚珠与败育胚珠ChSEP分布特征差异不明显。 结论 本研究在转录与蛋白水平上提供了ChSEP（Cor0054610.1）参与榛子胚珠发育调控的证据，为深入解析榛子胚珠发育机理提供了科学依据。 Abstract:Objective Normal ovule (kernel) filling is the basis of hazel kernel formation. Insufficient ovule filling can frequently lead to the shriveled kernels or empty nuts in hazel. SEP belonging to the E class MADS-box gene plays an important role in ovule development of plants. To investigate the important role of ChSEP in hazelnut ovule development, the key ChSEP genes involved in the development of hazelnut ovules were identified. Then, polyclonal antibody against ChSEP was prepared, followed by immunohistochemical analysis of ChSEP using developing and aborted ovules as study materials. The present study is beneficial for better understanding of ovule development mechanism regulated by SEP in hazelnut. Method All SEP gene family members in hazelnut genome were identified using genome and transcriptome data, and prokaryotic expression vector construction containing important SEP gene, gene expression, and polyclonal antibody preparation were carried out. Finally, important SEP involved in ovule development regulation was detected by immunohistochemistry. Result (1) Three ChSEP sequences were identified in the genome of hazelnut, including Cor0054610.1, Cor0008190.1 and Cor0119400.1. All SEP proteins contained SRF-TF and K-box motifs. Polyclonal antisera of Cor0054610.1 was prepared, and the molecular mass of polyclonal antibody E18305 and E18306 was about 45 kDa, and 1−2 ng antigen could be detected after 1∶1000 dilution. (2) Immunohistochemical analysis showed that ChSEP was expressed in the integument of both developing and abortive ovules. The expression of ChSEP in integuments was higher than that in cotyledons. There was no significant difference in the distribution of ChSEP between developing and abortive ovules. Conclusion This study provides evidence that ChSEP (Cor0054610.1) is involved in the regulation of hazelnut ovule development at both transcriptional and protein levels, and provides a scientific basis for better understanding of the ovule development mechanism in hazelnut. -
Key words:
- hazelnut /
- SEP /
- procaryotic expression /
- polyclonal antibody /
- immunohistochemistry
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图 1 拟南芥、葡萄、毛果杨与榛子SEP蛋白的系统发育分析
Figure 1. Phylogenetic analysis of A. thaliana, V. vinifera, P. persica and C. heterophylla SEP proteins
图 2 ChSEP基因家族多重序列比对（A）、保守结构域分布（B）与基因表达模式分析（C）
Figure 2. Multiple sequence alignment (A) and the conserve domain distribution (B) and gene expression model of ChSEP gene family (C)
图 3 ChSEP（Cor0054610.1）PCR扩增结果与蛋白表达纯化检测
A. PCR扩增结果：1. Marker；2. ChSEP（N-64-216）。B. 蛋白表达及可溶性检测：1. pGEX-4T-AB1空载诱导表达；2. 0.4 mg/mL BSA；3. Marker；4. 上清；5. 上清2；6. 包涵体2倍稀释（8 mol/L尿素溶解）；7. 包涵体10倍稀释（8 mol/L尿素溶解）。C. 纯化检测：1. Marker；2. 0.4 mg/mL 牛血清白蛋白； 3. 包涵体10倍稀释液（8 mol/L尿素溶解）；4. 包涵体5倍稀释液（8mol/L尿素溶解）。A, PCR amplification results: 1, marker; 2, ChSEP (N-64-216). B, protein expression and solubility detection: 1, pGEX-4T-AB1 empty-load-induced expression; 2, 0.4 mg/mL BSA; 3, marker; 4, supernatant; 5, supernatant 2; 6, two times dilution of inclusion bodies (dissolved in 8 mol/L urea); 7, ten times dilution of inclusion bodies (dissolved in 8 mol/L urea). C, purification detection:1, marker; 2, 0.4 mg/mL bovine serum albumin; 3, ten times dilution of inclusion bodies (dissolved in 8 mol/L urea); 4, five times dilution of inclusion bodies (dissolved in 8 mol/L urea).
Figure 3. ChSEP (Cor0054610.1) PCR amplification results and detection of protein expression and purification
图 4 ChSEP E18305（A）和E18306（B）抗体的Western Blot检测
泳道上方10 ng、5 ng、1 ng、500 pg分别表示该泳道中加有10 ng、5 ng、1 ng、500 pg ChSEP抗原。10 ng, 5 ng, 1 ng and 500 pg above the lane respectively indicate that 10 ng, 5 ng, 1 ng and 500 pg ChSEP antigen have been added to the lane.
Figure 4. Western blot detection of ChSEP E18305 (A) and E18306 (B) antibody
图 5 榛子正常发育与败育胚珠的免疫组织化学分析
A1与A2含正常发育与败育2个胚珠，A1为对照，一抗为清水，A2为处理，一抗为ChSEP抗体。B1与B2为正常发育胚珠，B1为对照，一抗为清水，B2为处理，一抗为ChSEP抗体。C1与C2为败育胚珠，C1为对照，一抗为清水，C2为处理，一抗为ChSEP抗体。ov. 败育胚珠；OV. 正常发育胚珠；Int. 珠被；F. 珠柄。A1 and A2 contain two ovules: normal development and abortive ovules. A1 is the control, the first antibody is distilled water, A2 is treatment, and the first antibody is ChSEP antibody; B1 and B2 are normal ovules, B1 is control, the first antibody is distilled water, B2 is treatment, and the first antibody is ChSEP antibody; C1 and C2 are aborted ovules, C1 is the control, the first antibody is distilled water, C2 is treatment, and the first antibody is ChSEP antibody. ov, abortive ovule; OV, normal development ovule. Int, integument; F, funicle.
Figure 5. Immunohistochemical analysis of normal development and abortive ovules of hazelnut
表 1 免疫流程
Table 1. Immunization process
过程
Process周期
Cycle剂量
Dose/mg弗氏佐剂
Freund’s adjuvant动物状态
Animal state第1次免疫 1st immunization 第1天 1st day 0.30 完全 Completely 良好 Good 第2 ~ 4免疫 2nd to 4th immunization 第12、26、40天 12th, 26th, 40th day 0.15 不完全 Incompletely 良好 Good 免疫动物采血
Blood collection from immunized animal第59天 59th day 采血正常
Blood collection is normal
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表 2 ChSEP（Cor0054610.1）抗血清ELISA效价检测
Table 2. ELISA titer detection of ChSEP (Cor0054610.1) antiserum
分组 Group 稀释比例 Dilution ratio OD450 （OD阳性−OD空白）/（OD阴性−OD空白）
（ODpositive−ODblank）/（ODnegative−ODblank）E18305 E18306 E18305 E18306 阳性对照 Positive control 1∶1 000 1.365 0 1.448 3 103.48 882.00 1∶4 000 1.230 8 1.384 7 92.25 842.25 1∶8 000 1.082 8 1.247 0 81.43 756.19 1∶16 000 0.902 9 1.142 2 67.38 690.06 1∶32 000 0.720 9 0.968 1 53.16 581.88 1∶64 000 0.533 9 0.767 2 704.86 33.19 1∶128 000 0.352 3 0.548 4 445.43 23.24 1∶256 000 0.2338 0.369 2 276.14 15.10 1∶512000 0.152 3 0.237 2 159.71 9.09 阴性对照 Negative control 1∶1 000 0.053 3 0.038 7 1∶64 000 0.041 2 0.059 1 空白对照 Blank control 0.040 5 0.037 1
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