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

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

摘要/Abstract

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。

关键词: 莱茵衣藻, 隐花色素, 蓝光, 突变株, 表型鉴定

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

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.

图/表 7

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

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

参考文献 45

[1]	崔红利, 陈军, 侯义龙, 等. 真核微藻蓝光受体及其功能研究进展[J]. 生物技术通报, 2017, 33(4): 51-62. doi: 10.13560/j.cnki.biotech.bull.1985.2017.04.007
	Cui HL, Chen J, Hou YL, et al. Research progress on blue-photoreceptors and its functions in eukaryotic microalgae[J]. Biotechnol Bull, 2017, 33(4): 51-62.
[2]	Butler WL, Norris KH, Siegelman HW, et al. Detection, assay, and preliminary purification of the pigment controlling photoresponsive development of plants[J]. Proc Natl Acad Sci USA, 1959, 45(12): 1703-1708. pmid: 16590561
[3]	Chen J, Kong Y, Wang Z, et al. Negative phototropism of Chlorophytum comosum roots and their mechanisms[J]. Hortic Plant J, 2015, 1(1): 55-60.
[4]	Chen J, Mo YW, Xu HW. Calcium signaling is involved in negative phototropism of rice seminal roots[J]. Rice Sci, 2014, 21(1): 39-46. doi: 10.1016/S1672-6308(13)60162-6
[5]	Galvão VC, Fankhauser C. Sensing the light environment in plants: photoreceptors and early signaling steps[J]. Curr Opin Neurobiol, 2015, 34: 46-53. doi: 10.1016/j.conb.2015.01.013 pmid: 25638281
[6]	Sakai T, Kagawa T, Kasahara M, et al. Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation[J]. Proc Natl Acad Sci USA, 2001, 98(12): 6969-6974. doi: 10.1073/pnas.101137598 pmid: 11371609
[7]	Garner WW, Allard HA. Effect of the relative length of day and night and other factors of the environment on growth and reproduction in plants[J]. Mon Wea Rev, 1920, 48(7): 415.
[8]	闫海芳, 周波, 李玉花. 光受体及光信号转导[J]. 植物学通报, 2004, 39(2): 235-246.
	Yan HF, Zhou B, Li YH. Photoreceptors and light signal transduction[J]. Chin Bull Bot, 2004, 39(2): 235-246.
[9]	Lepetit B, Dietzel L. Light signaling in photosynthetic eukaryotes with ‘green’ and ‘red’ chloroplasts[J]. Environ Exp Bot, 2015, 114: 30-47. doi: 10.1016/j.envexpbot.2014.07.007 URL
[10]	Cashmore AR. The cryptochrome family of photoreceptors[J]. Plant Cell Environ, 1997, 20(6): 764-767. doi: 10.1046/j.1365-3040.1997.d01-125.x URL
[11]	Lin CT, Shalitin D. Cryptochrome structure and signal transduction[J]. Annu Rev Plant Biol, 2003, 54: 469-496. pmid: 14503000
[12]	Sancar A. Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors[J]. Chem Rev, 2003, 103(6): 2203-2237. pmid: 12797829
[13]	Lin CT, Shalitin D. Cryptochrome structure and signal transduction[J]. Annu Rev Plant Biol, 2003, 54: 469-496. pmid: 14503000
[14]	Shalitin D, Yu XH, Maymon M, et al. Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1[J]. Plant Cell, 2003, 15(10): 2421-2429. doi: 10.1105/tpc.013011 pmid: 14523249
[15]	Kianianmomeni A, Hallmann A. Algal photoreceptors: in vivo functions and potential applications[J]. Planta, 2014, 239(1): 1-26. doi: 10.1007/s00425-013-1962-5 pmid: 24081482
[16]	孙燕, 许志茹. 植物的蓝光受体[J]. 植物生理学通讯, 2008, 44(1): 144-150.
	Sun Y, Xu ZR. Blue-light photoreceptors in plant[J]. Plant Physiol Commun, 2008, 44(1): 144-150.
[17]	Chaves I, Pokorny R, Byrdin M, et al. The cryptochromes: blue light photoreceptors in plants and animals[J]. Annu Rev Plant Biol, 2011, 62: 335-364. doi: 10.1146/annurev-arplant-042110-103759 pmid: 21526969
[18]	Coesel S, Mangogna M, Ishikawa T, et al. Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity[J]. EMBO Rep, 2009, 10(6): 655-661. doi: 10.1038/embor.2009.59 pmid: 19424294
[19]	Cock JM, Sterck L, Rouzé P, et al. The Ectocarpus genome and the independent evolution of multicellularity in brown algae[J]. Nature, 2010, 465(7298): 617-621. doi: 10.1038/nature09016 URL
[20]	Beel B, Müller N, Kottke T, et al. News about cryptochrome photoreceptors in algae[J]. Plant Signal Behav, 2013, 8(2): e22870. doi: 10.4161/psb.22870 URL
[21]	Heijde M, Zabulon G, Corellou F, et al. Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromes[J]. Plant Cell Environ, 2010, 33(10): 1614-1626. doi: 10.1111/j.1365-3040.2010.02168.x URL
[22]	Asimgil H, Kavakli IH. Purification and characterization of five members of photolyase/cryptochrome family from Cyanidioschyzon merolae[J]. Plant Sci, 2012, 185/186: 190-198. doi: 10.1016/j.plantsci.2011.10.005 URL
[23]	Oliveri P, Fortunato AE, Petrone L, et al. The cryptochrome/photolyase family in aquatic organisms[J]. Mar Genomics, 2014, 14: 23-37. doi: 10.1016/j.margen.2014.02.001 URL
[24]	Immeln D, Schlesinger R, Heberle J, et al. Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome[J]. J Biol Chem, 2007, 282(30): 21720-21728. doi: 10.1074/jbc.M700849200 URL
[25]	Immeln D, Pokorny R, Herman E, et al. Photoreaction of plant and DASH cryptochromes probed by infrared spectroscopy: the neutral radical state of flavoproteins[J]. J Phys Chem B, 2010, 114(51): 17155-17161. doi: 10.1021/jp1076388 URL
[26]	Beel B, Prager K, Spexard M, et al. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii[J]. Plant Cell, 2012, 24(7): 2992-3008. doi: 10.1105/tpc.112.098947 URL
[27]	Langenbacher T, Immeln D, Dick B, et al. Microsecond light-induced proton transfer to flavin in the blue light sensor plant cryptochrome[J]. J Am Chem Soc, 2009, 131(40): 14274-14280. doi: 10.1021/ja901628y pmid: 19754110
[28]	Li XB, Zhang R, Patena W, et al. An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[J]. Plant Cell, 2016, 28(2): 367-387. doi: 10.1105/tpc.15.00465 URL
[29]	Zhang R, Patena W, Armbruster U, et al. High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA[J]. Plant Cell, 2014, 26(4): 1398-1409. doi: 10.1105/tpc.114.124099 URL
[30]	Ahmed RA, He ML, Aftab RA, et al. Bioenergy application of Dunaliella salina SA 134 grown at various salinity levels for lipid production[J]. Sci Rep, 2017, 7(1): 8118. doi: 10.1038/s41598-017-07540-x URL
[31]	邵雪梅. 不同氮浓度对埃氏小球藻生长及油脂积累的影响[D]. 太谷: 山西农业大学, 2016.
	Shao XM. Effects of different nitrogen concentrations on growth and lipid accumulation of Chlorella emersionii[D]. Taigu: Shanxi Agricultural University, 2016.
[32]	Ninu L, Ahmad M, Miarelli C, et al. Cryptochrome 1 controls tomato development in response to blue light[J]. Plant J, 1999, 18(5): 551-556. doi: 10.1046/j.1365-313X.1999.00466.x URL
[33]	Imaizumi T, Kadota A, Hasebe M, et al. Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens[J]. Plant Cell, 2002, 14(2): 373-386. pmid: 11884681
[34]	Kanegae T, Wada M. Isolation and characterization of homologues of plant blue-light photoreceptor(cryptochrome)genes from the fern Adiantum capillus-veneris[J]. Mol Gen Genet, 1998, 259(4): 345-353. doi: 10.1007/s004380050821 URL
[35]	Das P, Lei W, Aziz SS, et al. Enhanced algae growth in both phototrophic and mixotrophic culture under blue light[J]. Bioresour Technol, 2011, 102(4): 3883-3887. doi: 10.1016/j.biortech.2010.11.102 URL
[36]	Gonçalves VD, Fagundes-Klen MR, Trigueros DEG, et al. Combination of light emitting diodes(LEDs)for photostimulation of carotenoids and chlorophylls synthesis in Tetradesmus sp[J]. Algal Res, 2019, 43: 101649. doi: 10.1016/j.algal.2019.101649 URL
[37]	Kuwano K, Abe N, Nishi Y, et al. Growth and cell cycle of Ulva compressa(Ulvophyceae)under LED illumination[J]. J Phycol, 2014, 50(4): 744-752. doi: 10.1111/jpy.12207 URL
[38]	沈银武, 朱运芝, 刘永定. 不同光质对中华植生藻的影响[J]. 水生生物学报, 1999, 23(3): 285-287.
	Shen YW, Zhu YZ, Liu YD. Effects of different light quality on richelia sinica[J]. Acta Hydrobiol Sin, 1999, 23(3): 285-287.
[39]	Im CS, Eberhard S, Huang KY, et al. Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii[J]. Plant J, 2006, 48(1): 1-16. doi: 10.1111/j.1365-313X.2006.02852.x URL
[40]	高松, 刘颖, 刘学娜, 等. 光质对大葱叶片碳氮代谢的影响[J]. 植物生理学报, 2020, 56(3): 565-572.
	Gao S, Liu Y, Liu XN, et al. Effects of light quality on carbon and nitrogen metabolism in leaves of Welsh onion(Allium fistulosum)[J]. Plant Physiol J, 2020, 56(3): 565-572.
[41]	李尚, 陶益, 刀国华, 等. 紫外线对再生水中斜生栅藻的生长抑制效果[J]. 环境工程, 2020, 38(10): 97-102, 113.
	Li S, Tao Y, Dao GH, et al. Growth suppression effect of UV-C irradiation on Scenedesmus obliquus in reclaimed water[J]. Environ Eng, 2020, 38(10): 97-102, 113.
[42]	周玉娇, 李亚军, 费小雯, 等. 小球藻紫外线诱变及高含油藻株筛选[J]. 热带作物学报, 2010, 31(12): 2124-2129.
	Zhou YJ, Li YJ, Fei XW, et al. UV-irradiation of Chlorella vulgaris and screening of petroliferous strains[J]. Chin J Trop Crops, 2010, 31(12): 2124-2129.
[43]	Atta M, Idris A, Bukhari A, et al. Intensity of blue LED light: a potential stimulus for biomass and lipid content in fresh water microalgae Chlorella vulgaris[J]. Bioresour Technol, 2013, 148: 373-378. doi: 10.1016/j.biortech.2013.08.162 URL
[44]	Fortunato AE, Jaubert M, Enomoto G, et al. Diatom phytochromes reveal the existence of far-red-light-based sensing in the ocean[J]. Plant Cell, 2016, 28(3): 616-628. doi: 10.1105/tpc.15.00928 URL
[45]	Ma RJ, Thomas-Hall SR, Chua ET, et al. Gene expression profiling of astaxanthin and fatty acid pathways in Haematococcus pluvialis in response to different LED lighting conditions[J]. Bioresour Technol, 2018, 250: 591-602. doi: 10.1016/j.biortech.2017.11.094 URL