Phenotypic Characterization of Blue Photoreceptor Plant Type Cryptochrome CRY Mutant in Chlamydomonas reinhardtii

doi:10.13560/j.cnki.biotech.bull.1985.2022-0611

Abstract

Abstract:

The phenotypic differences of Chlamydomonas reinhardtii wild strain（CC5325）and CRY mutant strain（crcry）under normal CK（Control）and Blue BL（Blue light）cultures were investigated. The PCR verification showed that the crcry mutant was inserted with paromomycin resistance gene AphVIII expression box in the coding region. AphVIII resistance gene was inserted into crcry mutant and successfully expressed in plate and liquid culture system with paromomycin as screening condition. Phenotypic identification showed that there were no significant differences in growth, pigment, photosynthesis and lipid synthesis between wild strain CC5325 and mutant strain crcry under CK culture condition. However, under BL condition, the growth of mutant crcry was significantly inhibited, and the color of algae liquid turned yellow. Chlorophyll a, chlorophyll b and total pigment contents of unit cells decreased significantly, while total carotenoid contents increased significantly. The photosynthetic system was severely suppressed and the total lipid content significantly reduced. In conclusion, plant cry is involved in the blue light response of C. reinhardtii.

Key words: Chlamydomonas reinhardtii, cryptochrome, blue light, mutant, phenotypic characterization

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.

Figures/Tables 7

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

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

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

Fig. 2 Effects of blue light on the growth of C. reinhardtii CC5325 and crcry CK: Normal light（white light）. BL: Blue light

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

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

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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