Introduction

‘Huangjinya’ belongs to light-sensitive etiolated tea species (Camellia sinensis), whose leaf color is regulated by light alone (Wang et al., 2008). Fan yangen et al. (2019, Shandong Agricultural University, pp.42-79) found that the contents of photosynthetic electron transport chain components of the yellowing and regreening leaves obtained from different shading treatments of ‘Huangjinya’ were significantly different from each other through comparative analysis of metabolomics and proteomics. The expression levels of the light-harvesting pigment antenna protein and most of the PSI and PSII complex subunit proteins in the yellowing leaf photosynthetic system were lower than those of the regreening leaf, only the LFNR and Fd proteins of electron transport chain and the PsbS protein of photosystem II (PSII) was up-regulated in yellowing leaves, and LFNR and PsbS were significantly up-regulated (Fan et al., 2019).

The leaf form of FNR (LFNR) is mainly responsible for transferring the electrons of reduced ferredoxin (Fd^red) to NADP⁺ for production of NADPH (Lintala et al., 2007). Marina et al. (2016) showed that the content and location of LFNR affected the content of superoxide anion in chloroplast, NADP (H) redox balance and glutathione redox state, thus regulating plant gene expression in response to adversity, such as high-light stress. Rodriguez et al. (2007) constructed transgenic tobacco (Nicotiana tabacum)plants expressing a pea (Pisum sativum) FNR in chloroplasts and they found that lines overexpressing the reductase showed augmented tolerance to photooxidative damage and redox-cycling oxidants. The distribution of LFNR on the chloroplast intima, stroma and thylakoid membrane showed a circadian rhythm: part of LFNR on the thylakoid membrane was released into the stroma in the daytime, and part of LFNR in the stroma was reattached to the membrane in the evening, and the amplitude of this dynamic change was directly related to the light intensity (Yang et al., 2014). At present, it has been found that the two membrane proteins Tic62 and TROL that anchor LFNR and an interaction protein LIR1 that responds to changes in light intensity are related to this process (Yang, 2015, Zhejiang University, pp.3-39; Hu, 2015, Zhejiang University, pp.6-32; Lin, 2016, Zhejiang University, pp.2-39).

In order to explore the role of LFNR in ‘Huangjinya’ leaf colour response to the light intensity change, this experiment took the new shoots of four-year-old ‘Huangjinya’ tea plant as the material, while using to the new shoots of ‘Shuchazao’ in Tea Plant Information Archive (TPIA) as the control, Cloning, bioinformatics analysis and protein subcellular localization analysis of LFNR gene were carried out respectively, the expression of the LFNR gene in the leaves of the ‘Huangjinya’ under different shades was detected by qRT-PCR. The purpose is to explore the relationship between the ‘Huangjinya’ LFNR species, the physicochemical properties of the coding protein, the subcellular localization, the gene expression level and light intensity, so as to provide experimental basis for further elucidation of the molecular mechanism of leaf yellows in the ‘Huangjinya’.

1 Results and Analysis

1.1 Cloning of CsLFNR1.1 and sequence analysis

The agarose gel electrophoresis showed that there was an obvious target gene around 1 095 bp (Figure 1). Specific primer PCR method was used to identify the positive clones,Sequencing was carried out in 10 tubes of bacterial liquid (5 tubes each of ‘Huangjinya’ and ‘Shuchazao’) sequencing, the sequencing results using DNAMAN and BioEdit software analysis, the results showed that 5 tubes microbial gene sequencing results were exactly the same, including 3 tube was derived from the ‘Huangjinya’ material, 2 tubes was derived from ‘Shuchazao’ material, named as CsLFNR1.1. The nucleotide sequence has been submitted to GenBank database and has been allocated the accession number MT311318. In addition, the gene sequence of 3 tubes (‘Huangjinya’ 1 tube and ‘Shuchazao’ 2 tubes) was different from that of CsLFNR1.1.However, due to the degeneracy of codons, the translation results of amino acids were the same. In addition, 1 tube of ‘Huangjinya’ bacteria solution gene had G base insertion at 1033bp and 1074bp, resulting in misreading of amino acid shift. 1 tube the amino acid sequence encoded by the gene of ‘Shuchazao’ was completely consistent with the LFNR provided by NCBI ‘Shuchazao’ genome.

The length of CsLFNR1.1 open reading frame (ORF) is 1 095bp, encoding 364 amino acid residues. By Nucleotide BLAST, it was found that the similarity between CsLFNR1.1 and NCBI LFNR gene sequence (XM_028233617.1) reached 99.63%, which was represented by CsLFNR1 in this article (Figure 2).

CsLFNR1.1 has four bases different from CsLFNR1, and there are three differences in the derived amino acid sequences.The first base A of the codon at 643bp becomes G, and the isoleucine (I) at position 215 of the corresponding protein amino acid sequence becomes valine (V). The third base A of the codon at 777bp becomes G, but the degeneracy of the codon don’t lead to the change of amino acid species. At 953bp, the second A of the codon becomes T, and the tyrosine (Y) at position 318 of the protein becomes phenylalanine (F); At 1066bp, the first base G of the codon becomes T, and the alanine (A) at 356 becomes serine (S).

1.2 Bioinformatics analysis of CsLFNR1.1gene

1.2.1 Analysis of amino acid composition and physicochemical properties of CsLFNR

Both CSLFNR1.1 and CsLFNR1 proteins are composed of 20 amino acids, with Lysine (10%) accounting for the largest proportion, Glycine, Leucine, and Valine accounting for 8% respectively, followed by Histidine (1%), which accounts for the least proportion (Table 1).

The molecular weights of CsLFNR1.1 and CsLFNR1 are 40.623 kDa and 40.637 kDa respectively, and the total number of atoms are 5 733 and 5 736. The instability index of the two proteins is less than 40 (when the instability index is less than 40, it means stability), and the total average hydrophobic index is less than 0, suggesting that the two proteins are stable hydrophilic proteins. The isoelectric points of the two proteins are both 8.86, and the basic amino acid residue base is higher than the acid amino acid residue base, which is the basic protein (Table 2).

1.2.2 Conserved domain analysis and active site prediction of CsLFNR

Based on the conserved domain analysis of NCBI CDD online database, it was found that the sequences of CsLFNR1.1 and CsLFNR1 from the 1st to the 364 amino acid regions all belong to the PLN03115 (Accession number: PLN03115) superfamily (Figure 3A). Motif Scan software was used to predict the active structural sites of CSLFNR1.1 and CsLFNR1 proteins (Figure 3B), and the predicted structural sites were consistent. The amino acids at position 85-207 were the FAD binding domain of ferridoxin reductase type (FAD_FR), and the amino acids at position 217-333 were the REDOX NAD binding domain (NAD_binding_1).

1.2.3 Signal peptide, transport peptide and transmembrane structure prediction of CsLFNR

SingalP 4.1 Server prediction results showed that there were no signal peptides in CsLFNR1 and CsLFNR1.1. ChloroP1.1 Server prediction results showed that both CsLFNR1 and CsLFNR1.1 contained a chloroplast transport peptide (cTP) with a length of 55 amino acid residues. The prediction results of TMHMM severv.2.0 online tool showed that there were no transmembrane spiral structure in both CsLFNR1 and CsLFNR1.1.

1.2.4 Prediction and analysis of secondary and tertiary structures of CsLFNR

Both CsLFNR1.1 and CsLFNR1 encoded amino acid sequences include leux-helix, woh-angle, extension chain and unregulated curling, and unregulated curling was the main component of the secondary structure, accounting for more than 40% of the total structure, and the lost-angle ratio is the least, accounting for 5.49% and 6.04% of the total structure of CsLFNR1.1 and CsLFNR1 respectively. CsLFNR1.1 and CsLFNR1 used the same template for the prediction of the tertiary structure, but they were slightly different in the prediction of the tertiary structure model (Table 3; Figure 4).

1.2.5 Evolutionary tree analysis and amino acid sequence alignment of CsLFNR

Protein BLAST homology query of CsLFNR1.1 encoded amino acid sequences in NCBI database showed that CsLFNR1.1 had a high homology with the LFNR amino acid sequences of several species.Through building evolutionary tree and comparison analysis of DNAMAN software, it was found that CsLFNR1.1, CsLFNR1, Actinidia chinensis (PSS36556.1) and Asparagus officinalis (XP_020248380.1) was in the same evolutionary branch that was more similar (Figure 5), and the similarity of amino acid sequence was 99.18%, 89.86% and 83.38% respectively (Figure 6).

1.3 Subcellular localization analysis of CsLFNR1.1

The constructed 35s :: CsLFNR1.1-GFP fusion tag vector (Figure 7) and the 35s :: GFP empty vector were transferred to Agrobacterium GV3101 and injected into tobacco(Nicotiana benthamiana) leaves for transient expression, observed by laser confocal microscopy (Figure 8): The GFP protein was distributed throughout the tobacco epidermal cells when expressed alone. The green fluorescence of the 35s :: GFP empty carrier protein under the excitation of 488nm blue light did not coincide with the red fluorescence of the chlorophyll in the tobacco epidermal cells. The luminescent part of the GFP fusion protein was in the chloroplast, and the green fluorescence emitted by it overlaps with the red fluorescence of chlorophyll to show yellow fluorescence.Therefore,CsLFNR1.1 gene encodes protein subcellular localization in chloroplast.

1.4 Expression analysis of CsLFNR1.1 gene in ‘Huangjinya’ leaves under different light intensity

The gene expression of CsLFNR1.1 in the ‘Huangjinya’ variety increased with the increase of light intensity. Among them, the CsLFNR1.1 gene expression levels of ‘Huangjinya’ leaves with no shading (H_S) and moderate shading (H_4W) were 1.82 times and 1.24 times higher than those of leaves with severe shading (H_1w) respectively. SPSS single factor ANOVA analysis showed that there were significant differences between H_1w, H_4W and H_S (Figure 9).

Note: Different lowercase letters represent significant differences in relative expression of CsLFNR1.1 among different light density (LSD Duncan test, p＜0.05)

2 Discussion

This study is based on tea tree transcriptome sequencing results,the experiment used ‘Huangjinya’ and ‘Shuchazao’ tea tree leaves separately as material to clone CsLFNR gene,obtained the same sequence in the end,named CsLFNR1.1.The results showed that the CsLFNR1.1 with CsLFNR1 (NCBI reference number: XM_028233617.1) gene had four different bases, resulted in CsLFNR1 with CsLFNR1.1 three different amino acids: I₂₁₅→V₂₁₅, Y₃₁₈→F₃₁₈, A₃₅₆→S₃₅₆. According to bioinformatics analysis,the amino acid sequence similarity of CsLFNR1.1 and CsLFNR1 was 99.18%, the sequences from 1 to 364 amino acid regions all belonged to the conserved sequence of PLN03115(Accession number: PLN03115)  superfamily protein, with exactly the same FAD binding domain (amino acids at positions 85 to 207), but the NAD binding domain (amino acids at positions 217 to 333) existed differences in amino acid (318^th):CsLFNR1 for tyrosine (Y₃₁₈), CsLFNR1.1 for phenylalanine (F₃₁₈). Therefore, whether there is any difference between CsLFNR1.1 and CsLFNR1 in enzyme and substrate affinity needs to be further studied.

Although CsLFNR1.1 and CsLFNR1 had four bases of difference ,resulting changes in three loci related protein amino acid, but CsLFNR1.1’s conservative sequence, active site structure, tertiary structure prediction template and subcellular localization were the same as CsLFNR1,It’s speculated that CsLFNR1.1 and CsLFNR1 were homologous genes. It’s worth noting that the CsLFNR1.1 gene can be cloned from the yellow variety ‘Huangjinya’ and the green variety ‘Shuchazao’ at the same time, it’s supposed that the amino acid difference between CsLFNR1.1 and CsLFNR1 wasn’t the direct cause of the yellow color of the new shoots of the ‘Huangjinya’ tea variety under the high light.

Analysis of CsLFNR1.1 expression in leaves of ‘Huangjinya’ with different shading degrees showed that CsLFNR1.1 gene expression was positively correlated with light intensity, which was consistent with proteomic research results of Fan yangen et al.(2019, Shandong Agricultural University, pp.42-79):compared with‘Huangjinya’ leaves under 80% shading conditions, FNR protein content in ‘Huangjinya’ leaves under natural light was significantly increased. The above results showed that the expression of CsLFNR1.1 gene was regulated by light and was correlated with the changes of leaf color of ‘Huangjinya’.

Although the light-regulated circadian mechanism of LFNR distribution in the chloroplast intima, stroma and thylakoid membrane had been investigated (Yang et al., 2014), the light-regulated pathway of LFNR gene expression in tea tree is still not clear, which needs to be further explored.

3 Materials and Methods

3.1Materials and chemicals

3.1.1 Materials

Gene cloning materials: Four-year-old tea plants growing in the experimental field of Shandong Agricultural University (SDAU, N361.8’, E117.1’), Tai-an city, China, were used for this study. Samples were flash frozen with liquid nitrogen and stored at -80°C.

Gene expression material: Second leaves under new tip buds of ‘Huangjinya’ with different shade degrees: double needle 6 pins mesh (average light intensity of about 180 μmol photons·m⁻²·s^-1, number H_1w), Single shade 6 pins mesh (average light intensity of about 720 μmol photons·m⁻²·s^-1, number H_4w) and no shade processing blade (average light intensity about 1 620 μmol photons·m−2·s-1, number H_S) as materials. Samples were flash frozen with liquid nitrogen and stored at -80°C.

3.1.2 Chemicals

Gene cloning reagents: 6×DNA Loading Buffer,2×RNA Loading Buffer, LB Liquid medium, solid medium dry powder、Ampicillin Sodium salt were from solarbio science﹠technology co., ltd. (Beijing, China). Biowestregular agarose G-10(Hong Kong, China).EB Dye substitute,β-mercaptoethanol (BME)were bought from SanGon Biotech Co., Ltd. (Shanghai, China). TransStart® FastPfu Fly DNA Polymerase Gene amplification kit, pEASY®-Blunt Simple Cloning Vector test kit were purchased from TranGen Biotech Co., Ltd. (Beijing, China), Common agarose gel DNA recovery kit, 2×Pfu PCR MasterMix (KP201) were purchased from TIANGEN BIOTECH Co., Ltd. (Beijing, China).ddH₂O(Thermo GenPure UV/UF, USA).DH5αwas bought from Biomed Biotech Co., Ltd. (Beijing, China).Yeast Extract, Tryptone were purchased from Pronade technology co., LTD.

Subcellular localization reagents: FastDigest SmaI enzyme digestion kitwas bought from Thermo Fisher Scientific co., LTD. ClonExpress Ultra One Step Cloning Kit was bought from Vazyme co., LTD (Nanjing, China).pRI101-AN-GFP expression vector.GV3101(pSoup-P19) Electro was bought from Angyu Biotech co., LTD (ShangHai, China).1/2MS medium, sucrose, ethanol were bought from Kaitong Chemicals co., LTD (Tianjing, China).Kanamycin mono sulfate, Rifampicin antibiotic, MES, AS, DMSO were from Beijing solarbio science﹠technology co., ltd. (Beijing, China).

qRT-PCR reagents: cDNA first chain synthesis premix kit was bought from TranGen Biotech Co., Ltd. (Beijing, China). FastPure Plant Total RNA Isolation Kit (Polysaccharides& Polyphenolics-rich), HiScript Q Select RT SuperMix for qPCR, ChamQ Universal SYBR qPCR Master Mix were bought from Vazyme co., LTD (Nanjing, China).

3.1.3 Instruments and equipment

Nucleic Acid concentration Meter (GeneQuant Pro), Electrophoresis system(Biorad), Ultraviolet gel imager (Nanjing, China),Sterilization pot (Panasonic ML5-3751L),Pipetting gun (Biohit), Centrifuge (Sigma3-18 k),T100 Thermal Cycler, Digital display thermostatic water bath pot, LightCycler 480 system, Laser scanning confocal microscope LSM880.

3.2 Total RNA extraction and gene cloning

Total RNA was extracted from the tea leaves using an RNAprep Pure Plant Kit (Vazyme, Nanjing, China) as per the manufacturer’s protocol. Then, 1% agarose gel electrophoresis was used to check the quality and purity of the total RNA, and a gel imaging system (GelDoc-1T2 315, BioSpectrum, USA) was used to observe the electrophoretic strip. First strand cDNA was synthesized using a reverse transcription kit. According to the TPIA (webpage: http://tpia.teaplant.org) ‘Shuchazao’ tea tree genome LFNR gene (TEA032600) sequence to design the upstream and downstream primers (Table 4). After cloning product recovery, connecting with pEASY®-blunt Simple cloning vector, and positive monoclonal colonies were selected for sequencing after transformation into E. coli DH5α.

3.3 Bioinformatics analysis

Protoparam (http://web.expasy.org/protparam) and Protoscale (http://web.expasy.org/protscale) were used to analyze the physicochemical properties and hydrophilicity/hydrophobicity of the encoded amino acid sequence. we used SINGLP 4.1 Server, Chlorop 1.1 Server, TMHMM server2.0 to analyze signal peptides encode amino acid sequences, chloroplast transport peptides and transmembrane structures. The sopma  and Swiss-Model  were used to predict the secondary and tertiary structures of the encoded protein. CDD (Conserved Domain Database) of NCBI was used to analyze the protein conserved domain. Motif Scan software was used to predict the active sites of proteins. The homologous gene coding protein was obtained by blastp, the phylogenetic tree was constructed by MEGA7.0 software, and homologous sequence alignment was performed by DNAMAN software.

3.4 Protein subcellular localization

Download the primer design software CEDesign (http://www.vazyme.com) and generate homologous recombination amplification primers of inserted target fragments (Table 5).

The plasmid extracted from CsLFNR1.1 bacterial liquid with correct sequencing is used as a template, CsLFNR1.1-CZF and CsLFNR1.1-CZR are used as primers to clone and recover the target fragment, and the extracted plasmid and pRI101-GFP vector are subjected to FastDigest SmaI single enzyme digestion at the same time. The ClonExpress®Ultra One Step Cloning Kit (Vazyme, Nanjing, China) is adopted to carry out single fragment recombination reaction, the recovered target fragment is connected to pRI101-GFP vector, the connection product is transformed into Escherichia coli, and positive monoclonal sequencing is carried out. The 35s:: CsLFNR1.1-GFP plasmid successfully constructed from correctly sequenced E.coli shaking bacteria was extracted and transformed into Agrobacterium GV3101 by liquid nitrogen quick freezing method. Positive colonies were identified by PCR. The subcellular localization method of Agrobacterium infecting tobacco (Nicotiana benthamiana) epidermal cells was operated with reference to Xu and Kong (2017).

3.5 Quantitative real-time polymerase chain reaction

Total RNA was extracted from the tea leaves using an RNAprep Pure Plant Kit (Vazyme, Nanjing, China) as per the manufacturer’s protocol. The cDNA obtained through reverse transcription was operated according to the instructions of ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The gene primers for qRT-PCR in this experiment were designed by Primer 5 software and were shown in Table 6. β-actin (Sun et al., 2010) was chosen as an internal reference control. A real-time PCR detection system (LightCycler 480 system, Roche Applied Science, Mannheim, Germany) was used as the fluorescence quantitative polymerase chain reaction instrument. Relative quantification values were determined using the 2 ^–ΔΔCt method (Livak and Schmittgen, 2001).

3.6 Statistical analysis

Using Excel 2016 software to statistical analysis of data, using SPSS 19.0 software for difference significance test (α=0.05), the Microsoft Office Visio software was used to draw.

Authors’ contributions

ZX, ZL and LM are the experimental design and executors of this research; ZX completed the data collation and writing of the first draft of the paper; LM, FY, TY, WH participated in the experimental design and analysis of the results of the experiment; ZL is the planner and person in charge of the project, guiding experiment design, data analysis, and writing and revising papers. All authors read and agreed to the final manuscript.

Acknowledgements

This work was supported by the Found of Shandong ‘DoubleTops’ Program (SYL2017YY03).

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