生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 243-253.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0611
李旺宁1(), 张豪杰1, 李亚男1, 梁梦静1, 季春丽1, 张春辉1, 李润植1, 崔玉琳2, 秦松2, 崔红利1()
收稿日期:
2022-05-18
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
李旺宁,女,硕士研究生,研究方向:微藻生物技术;E-mail: 基金资助:
LI Wang-ning1(), ZHANG Hao-jie1, LI Ya-nan1, LIANG Meng-jing1, JI Chun-li1, Zhang Chun-hui1, LI Run-zhi1, CUI Yu-lin2, QIN Song2, CUI Hong-li1()
Received:
2022-05-18
Published:
2023-02-26
Online:
2023-03-07
摘要:
以莱茵衣藻(Chlamydomonas reinhardtii)野生株(CC5325)和植物类型CRY突变株(crcry)为研究对象,探究其在正常光照CK(Control)与蓝光BL(Blue light)培养条件下的表型差异。PCR检测表明,crcry突变株在编码区插入含有巴龙霉素抗性基因AphVIII的表达盒。以巴龙霉素为筛选条件的平板和液体培养体系进一步验证,crcry突变株中插入了AphVIII抗性基因并成功表达。表型鉴定表明,在CK培养条件下,野生株CC5325和突变株crcry在生长、色素、光合及油脂合成等方面没有显著差异;但是在BL条件下,突变株crcry的生长受到明显抑制,藻液颜色变黄;单位细胞叶绿素a、叶绿素b及总色素含量显著下降,相反,总类胡萝卜素含量明显增高;光合系统受到严重抑制,以及总脂含量显著降低。植物类型cry参与莱茵衣藻蓝光响应过程。
李旺宁, 张豪杰, 李亚男, 梁梦静, 季春丽, 张春辉, 李润植, 崔玉琳, 秦松, 崔红利. 莱茵衣藻蓝光受体植物类型隐花色素CRY突变体的表型鉴定[J]. 生物技术通报, 2023, 39(2): 243-253.
LI Wang-ning, ZHANG Hao-jie, LI Ya-nan, LIANG Meng-jing, JI Chun-li, Zhang Chun-hui, LI Run-zhi, CUI Yu-lin, QIN Song, CUI Hong-li. Phenotypic Characterization of Blue Photoreceptor Plant Type Cryptochrome CRY Mutant in Chlamydomonas reinhardtii[J]. Biotechnology Bulletin, 2023, 39(2): 243-253.
引物名称Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
Actin-F | AGAAGGACTCGTACGTTGGC |
Actin-R | CCAGAGTCCAGCACGATACC |
cry-F | AGCACCCAATGTGATACCCG |
cry-R | CATACAGCATGTCGCCGTTG |
Cassette-C1 | ATACTGCATGTAATGGCCAGG |
Cassette-C2 | GCGGCAGAATAGTCGCGTAT |
表1 引物信息
Table 1 Primers information
引物名称Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
Actin-F | AGAAGGACTCGTACGTTGGC |
Actin-R | CCAGAGTCCAGCACGATACC |
cry-F | AGCACCCAATGTGATACCCG |
cry-R | CATACAGCATGTCGCCGTTG |
Cassette-C1 | ATACTGCATGTAATGGCCAGG |
Cassette-C2 | GCGGCAGAATAGTCGCGTAT |
试剂Reagent | 用量Dosage/μL |
---|---|
TB Green Premix Ex Taq II(TliRNaseH Plus)(2×) | 5 |
正向引物 | 0.25 |
反向引物 | 0.25 |
DNA模板 | 0.5 |
双蒸水 | 4 |
反应总体系 | 10 |
表2 PCR反应体系
Table 2 PCR reaction system
试剂Reagent | 用量Dosage/μL |
---|---|
TB Green Premix Ex Taq II(TliRNaseH Plus)(2×) | 5 |
正向引物 | 0.25 |
反向引物 | 0.25 |
DNA模板 | 0.5 |
双蒸水 | 4 |
反应总体系 | 10 |
图1 莱茵衣藻crcry突变体的鉴定 a:插入盒示意图;b:插入位点DNA水平的PCR验证;c:插入位点RNA水平的PCR验证;d:突变株固体平板筛选;e:突变株液体筛选。P:巴龙霉素
Fig.1 Identification of C. Reinhardtii crcry mutant a: Schematic map of the inserted cassette. b: PCR confirmation of insertion site DNA level. c: PCR validation of RNA levels at insertion sites. d: Plate screening of mutant strains. e: Liquid screening of mutant strains. P: Paromomycin
图2 蓝光对莱茵衣藻CC5325和crcry生长的影响 CK:正常光(白光);BL:蓝光
Fig. 2 Effects of blue light on the growth of C. reinhardtii CC5325 and crcry CK: Normal light(white light). BL: Blue light
图3 蓝光对莱茵衣藻CC5325和crcry单位细胞色素的影响 a:单个细胞叶绿素a的含量; b:单个细胞叶绿素b的含量; c:单个细胞叶绿素c的含量; d:单个细胞叶绿素d的含量; e:单个细胞叶绿素e的含量; f:单个细胞叶绿素f的含量
Fig. 3 Effects of blue light on the single cell pigments of C. reinhardtii CC5325 and crcry a: Chlorophyll a content in a single cell. b: Chlorophyll b content in a single cell. c: Content of chlorophyll c in a single cell. d: Content of chlorophyll d in a single cell. e: Chlorophyll e content in a single cell. f: Chlorophyll f content in a single cell
图4 蓝光对莱茵衣藻CC5325和crcry光合作用的影响 a:光系统II实际光化学效率;b:Fv/Fm 最大光化学效率;c:相对电子传递速率;d:非光化学淬灭系数;e:光化学淬灭系数
Fig. 4 Effects of blue light on the photosynthesis of C. Reinhardtii CC5325 and crcry a: Actual photochemical efficiency of photosystem II. b: Maximum photochemical efficiency of Fv/Fm. c: Relative electron transfer rate. d: Non-photochemical quenching coefficient. e: Photochemical quenching coefficient
图5 蓝光对莱茵衣藻CC5325和crcry总脂的影响 不同小写字母表示在P<0.05水平差异显著
Fig. 5 Effects of blue light on the total lipid of C. Reinh-ardtii CC5325 and crcry Different lowercase letters indicate significant difference at 0.05 level
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