Transcriptome Analysis of Photosynthetic Characteristics was Induced by Low Temperature Stress in Brassica napus L.

Photosynthetic Abstract Background RNA Sequencing (RNA-Seq) technique could be utilized to compare the transcription groups of two different cold-resistant rapeseed leaves responding to low temperature at the seedling stage, analyze the photosynthetic characteristics of rapeseed subjected to low temperature stress, and identify the related genes for low temperature induction in rapeseed leaves. Results Using cold-tolerant variety 17NS and sensitive variety NF24 as experimental materials, carrying out RNA-Seq analysis by photosynthetic parameter determination and Illumina HiSeqTM platform. and screen out the KEGG significant enrichment pathway related to photosynthetic characteristics under low temperature stress. Differential Expressed Genes (DEGs) were used for real-time PCR to verify the reliability of RNA-Seq results. The results showed that the response of Brassica napus L. to low temperature stress mainly was achieved by inhibiting photosynthesis, the cold-tolerant variety 17NS had a strong ability to maintain membrane system stability and structural integrity after 24 h of low temperature stress, while the sensitive variety NF24 photosynthesis was significantly inhibited. Two pathways of Photosynthesis and Photosynthesis-antennas, which were significantly correlated with photosynthetic characteristics and low temperature stress were screened by KEGG enrichment. The results of DEGs indicated that 64 differentially expressed genes in these two pathways were induced by low temperature stress, and 8 of them were up-regulated expression and 56 of them were down-regulated expression. The expression pattern of DEGs was consistent with the results of RNA-Seq analysis by qRT-PCR detection and confirmed the reliability of RNA-Seq results. Conclusion Our study analysis and identified 17 low-temperature-induced photosynthetic-related candidate genes in Brassica napus L., and the GO and KEGG metabolic pathways clarified the molecular function of differentially expressed genes.

temperature inhibits plant photosynthetic pigment content and physiological processes such as photosynthesis [14][15][16][17]. Photosynthesis is the main pathway for plant vegetative organs, which can use light energy to synthesize organic compounds from carbon dioxide and water, from light-dependent parts (photoreaction) and light independence part (dark reaction, carbon fixation) composition. Low temperature stress can cause a decrease in photosynthetic parameters such as agronomic traits, photosynthetic rate, transpiration rate, leaf stomatal conductance and intercellular CO 2 concentration [18,19]. Chloroplasts, as the main organelles for photosynthesis of plants, are also the most sensitive target organs for stress such as low temperature [20]. After chilling stress, the morphological structure of chloroplasts changes significantly. The chlorophyll content is reduced to reduce the light-capturing ability of leaves [21], and the grainy lamella are reduced or stress was revealed were analyzed,.

Responses of plant morphology and leaf development to low temperature stress
Observation on the morphological development of plants under low temperature stress The Brassica napus L. was treated at low temperature for 24 h at 4 °C (Fig.1). The morphological changes of the plants showed that the leaves of 17NS and NF24 had wilting and sagging, of which the old leaves of sensitive NF24 were obviously wilted, the new were leaves slightly wilted, and the old leaves of cold tolerant 17NS were wilted while the new leaves barely changed. The two varieties after low temperature treatment were restored at room temperature for 24 hours. It was found that the old leaves of the sensitive variety NF24 all died, the new leaves returned to life, and the cold-tolerant variety 17NS recovered after 24 hours, the new leaves and the old leaves are green and revitalized. The results showed that the new leaves showed strong tolerance and adaptability in response to lo w temperature stress and the leaves of the cold-resistant variety 17NS have strong frost resistance under low temperature stress.
Observation chloroplast morphological under low temperature stress by transmission electron microscopy Transmission electron microscopy observation of rapeseed leaves under low temperature stress showed that low temperature stress had a great influence on the structure and quantity of organelles in leaf cells, which were manifested in the morphological structure of chloroplasts, the number of hypersalotic particles and starch granules were changed.
The varieties with different cold resistance have different degrees of changes in organelles. After 24 hours of cold tolerance of 17NS cold-resistant varieties, the granum were dispersed, the chloroplasts shrank, the volume became smaller, and the starch granules became smaller ( Fig.2 -A1). Compared with the control (Fig.2-B1), the number of starch granules increased. There are up to 3 starch granules in the chloroplast, and the number of starvation granules increases (Fig.2-B2), the number and morphology of chloroplasts are not obvious (Fig.2-B3).In the sensitive variety NF24, the control ( Fig. 2-C1) starch granules were faintly visible, disappeared after low temperature stress, and the thylakoid layer was significantly reduced (Fig.22-D1). The chloroplast changes from a spindle shape close to the cell wall to a paramecium shape far away from the cell wall, the number of mitochondria increased significantly ( Fig.2-D2), and the number of chloroplasts in individual cells decreased (Fig.2-D3).It indicated that the varieties with different cold resistance had different responses to chloroplast photosynthesis and respiration after low temperature stress.

Response of Physiology and Biochemistry of Brassica napus L. to Low Temperature Stress
Response of cell membrane of Brassica napus L. to low temperature stress When the plant tissue is damaged by the stress, the low temperature stress firstly damages the membrane system of the cell, the membrane function is impaired or the structure is destroyed, and the permeability is increased, the water-soluble substances in the cells, including the electrolyte, will have different degrees of extravasation. The relative conductivity measurement showed that the relative conductivity of the coldtolerant variety 17NS and the sensitive variety NF24 was almost the same before the low temperature stress. After the low temperature stress, the relative conductivity increased significantly (Fig.3). The relative conductivity of the sensitive variety NF24 increased sharply. It is 0.869 ms/cm 3 , which is larger than the rising value of 17NS (0.760 ms/cm 3 indicating that the electrolyte of the cell membrane of Brassica napus L. has a large amount of extravasation after low temperature damaging. Sensitive varieties have large electrolyte exosmosis, poor cold tolerance and adaptability while cold resistant variety have strong ability to maintain the relative stability and structural integrity of the cell membrane system, and better resistance to low temperature damage.

Effects of low temperature stress on photosynthetic parameter
The photosynthetic parameters of 0~24h under low temperature stress showed that the low temperature significantly reduced the net photosynthetic rate (Pn), stomatal more unstable than that of cold resistant cultivars at the low temperature stress. The Ci of the cold-tolerant variety 17NS and the cold-sensitive variety NF24 were first decreased and then increased. After 24 hours, the Ci increased by 16.29% and 20.01%, respectively. Therefore, low temperature significantly affects photosynthesis and stomatal gas exchange, and has a more greater impact on Tr, Gs and Pn of cold sensitive varieties.
Effect of low temperature on chlorophyll content of Brassica napus L It can be seen from with low temperature stress, and the content of carotenoids increased, and the decrease of chlorophyll content of cold resistant varieties was smaller than that of sensitive varieties, indicating that the cold resistant varieties had stronger resistance after low temperature stress. The strong photosynthetic capacity of the cold resistant variety is conducive to maintaining the normal growth of seedlings for adversity injury.
Screening of differentially expressed genes in Brassica napus L. under low temperature stress According to the transcriptome data of Brassica napus L. under low temperature stress, the difference gene expression was analyzed by using edgeR software. The differential gene was screened by FDR and log2FC. The screening criteria were FDR<0.05 and |log2FC|>1. Among the different cold cultivars, NF24t0-17NSt0 was paired to obtain 18978 genetic difference genes (Table 1.). NF24t-17NSt paired to obtain 22019 differential genes, and the genetic background difference was removed to obtain significant differences in low temperature induction. A total of 3041 genes were expressed (739 differential genes up-regulated expression and 2302 differential genes down-regulated expression).

KEGG pathway analysis
By KEGG Pathway analysis of differential genes, the pathway with Qvalue ≤ 0.05 was chosen as the pathway for differential gene enrichment. The results showed that in all differentially expressed genes, after 24 h of low temperature stress, 4229 differentially expressed genes of NF24t0-17NSt0 and 4675 differentially expressed genes of NF24t-17NSt were enriched in 131 and 132 pathways, respectively. Twenty of the most significant pathways were screened by P<0.05 (Fig.6), and Ribosome and Metabolic pathway were the most significant pathways before and after low temperature stress, respectively.
Light and regulation related differential expression gene analysis The analysis of pathways related to photosynthetic characteristics after low temperature stress revealed that Photosynthesis and Photosynthesis-antenna proteins are significant difference in energy metabolism, of which Photosynthesis-antennas were ranked in the top 20 in the NF24 t-vs-17NS t comparison group (Fig.6). Using Venny online software (http://bioinfogp.cnb.csic.es/tools/venny/) to analyze the DEGs(Differentially Expressed Genes) of different varieties under low temperature stress (Fig. 7), it was found that the

Go function classification of specific expression genes
Through the function annotation ( Fig.11) of Go function, it is found that 64 specific DEGs related to photosynthetic regulation in the differentially expressed genes under low temperature stress are mostly annotated into cell component processes and biological processes, and the number of differentially expressed genes are least in molecular function. It indicated that low temperature stress affected the growth and development of Brassica napus L. Among them are mainly cellular processes, metabolic processes, cells, cellular components, organelles, binding, catalytic activity and transport activity.
qRT-PCR validation of specific expression genes The 64 low-temperature-induced specific DEGs ( Table 2)

Conclusions
Through the analysis of the photosynthetic parameters of Brassica napus L. under low temperature, the results showed that the photosynthesis was inhibition at low temperature stress, and the strong cold resistance varieties is suppressed to a lesser degree. By RNA -Seq analysis found that the response of low temperature stress significantly photosynthesis pathway for Photosynthesis and Photosynthesis-antenna proteins, got 64 significantly DEGs. By analyzing qPCR, verified the reliability of the transcriptome and determine the 17 low temperature response of photosynthesis regulation genes.

NS, a strong cold winter variety of Brassica napus strains and professor Liu of Gansu
Agricultural University breeding from "Tiangan" of Brassica napus L. as the female parent and "Longyou 7" of Brassica rapa L.as the male parent to selected homozygous line, and the cold sensitive material NF24 is an inbred line of Brassica napus L. which both have been undertook the formal identification by professor Liu in my study.

The aim
In order to reveal the molecular mechanism of rapeseed's adaptation to low temperature stress, RNA Sequencing (RNA-seq) technique is utilized to compare the transcription groups of two different cold-resistant rapeseed leaves responding to low temperature at the seedling stage, analyze the photosynthetic characteristics of rapeseed subjected to low temperature stress, and identify the related genes for low temperature induction in rapeseed leaves, by the determination of photosynthetic parameters and the RNS-seq analysis of Illumina HiSeqTM platform, the KEGG enrichment pathway associated with photosynthetic characteristics was identified under low temperature stress, and the (DEGs) in the significant enrichment pathway were Expressed by real-time quantitative PCR, to verify the reliability of the RNA-Seq results.

The design
By pot experiments, the seeds with the same clean and uniform size were placed in a petri dish with two layers of filter paper as the germination bed, cultured in an artificial climate chamber (6000 lux light 25 ° C / 14 h and 20 ° C / 10 h) , and then sown on mix nutrient matrix and vermiculite in a 3:1 pot after seed germination, artificial light incubator culture, light 25 ° C / 14 h and 20 ° C / 10 h, relative humidity 40%. When the seedlings grow to 5~6 leaf stage, the low temperature -4 °C light incubator starts low temperature treatment for 0h and 24h, two pots of which are used for photographing, two pots are used for photometry, and the other two are used for sampling. Wearing a mask and sterile enzyme-free gloves collects photographs and measures photosynthetic parameter, and liquid nitrogen quick freeze the newly-fully-expanded leaves after rinsing with sterilized up-water and stored in a -70°C ultra-low temperature freezer for determination of various indicators.

Determination of photosynthesis index
Six pieces leaves without shade, no damage, no pests, and good growth of the fourth functional leaf were selected for each material, and the small-label was placed. According to the method of Liu Zigang[32], the LI-6400 portable photosynthetic apparatus produced in the United States was used. The changes of photosynthetic gas exchange parameters (Gs, Ci, Tr, Pn) of new leaves that were fully developed at 0h, 12h and 24h after low temperature treatment were repeated three times, setting the light intensity to 1200μmol/m 2 , and the indoor temperature of the leaves was 20°C. .

Determination of chlorophyll content and relative conductivit
The chlorophyll content and relative conductivity were measured according to the scholar[33].

Transmission electron microscope
On the new leaves which were completely unfolded at 0 h and 24 h after low temperature treatment, a rectangular blade of 2 mm 2 was cut out with a blade carefully, and placed in a fixing solution containing 2 ml of 3% glutaraldehyde. According to the scanning procedure of electron microscopy, PB was rinsed three times, 15 min/time, hungry acid was fixed at 4 °C for 2 h, and then rinsed with PB three times, 15 min/time, according to 50%, 70%, 80%, 90%, 100%, acetone I and Acetone II gradient dehydration for 10 min each time. Acetone and resin embedding agent were embedded in an oven at 35 ° C for 12 h, and then transferred to an oven at 60 ° C until the material was completely polymerized. With the ultramicrotome, cutting into slices and fixing on a copper grid with 2% uranyl acetate and lead citrate for double staining and photographs were taken with a Hitachi JEM-1230 projection electron microscope.

Analysis of transcriptome data
The transcriptome analysis was carried out using cold-tolerant variety 17NS and lowtemperature sensitive variety NF24. The low temperature stress of -4 °C was used as the control, and the low temperature stress of -4 °C was used as the treatment for 24 hours.
Two varieties were treated at two different times, a total of four samples. Total RNA was extracted from the sample by the company named GENE DENOVO, the mRNA was enriched with magnetic beads with Oligo (dT), and the fragmentation buffer was added to the obtained mRNA to make a fragment. As a short fragment, the first strand of cDNA was synthesized with random hexamers using the mRNA after fragmentation, and the second strand of cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I, after QiaQuick The PCR kit was purified and eluted with EB buffer, the end of the repair, base A was added, the sequencing linker was added, and the fragment of interest was recovered by agarose gel electrophoresis, and PCR amplification was performed to complete the whole library preparation work. A good library was sequenced using Illumina HiSeq TM . After filtering the off-data, the clean data is compared, the reads are aligned to the reference genome, and the transcripts are assembled using Cufflinks to obtain known transcripts and new transcripts. Then, the obtained gene was analyzed and statistically analyzed, and differential expression analysis and functional enrichment analysis were performed. In addition, structural analysis of genes, including genetic structure optimization, variable shear, and the like.
Total RNA extraction from leaves RNA extraction The total RNA of 17NS-0h, 17NS-24h, NF24-0h and NF24-24h was extracted according to the instructions of Tiangen kit (DP419). The reverse transcription was reversed according to the TaKaRa cDNA First-strand Synthesis Kit (RR036A). Singlestranded cDNA was obtained by transcription, and the concentration was measured by Bio Mater 5 ultra-micro UV spectrophotometer, and then stored in a refrigerator at -80 ° C for use.

Real time PCR
Using the PrimeScript RT reagent Kit (Dalian Bioengineering Co., Ltd., Dalian), reverse transcription kit to reverse transcribe the RNA extracted from the previous step to synthesize the first strand of cDNA.According to the transcriptome data, the reverse transcription product was diluted 100-fold as a template, primers were designed with Primer Premier 5.0 (Table 3), the internal reference was Bnactin, and the primer was synthesized by Shanghai Biotech Co., Ltd., using SYBR Premix Ex Taq (Bao Bioengineering Co., Ltd., Dalian) Quantitative kit, two-step amplification, each reaction is set to 3 repeats, the reaction system and procedures are shown in Table 4, according to the amplification efficiency E of the gene, the correction 2 -△△Ct is calculated as (1+E) -△△Ct relative quantification As a result, statistical analysis was performed using Excel 2010 and SPSS 22.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests      The influence of low temperature to conductivity in rapeseed leaves. Note: The figure with P<0.01 as highly significant, marking "A, B or C", as the same below. Photosyntheticrate (μmol/m2/s).

Figure 5
The influence of low temperature to Chlorophyll content in rapeseed leaves Branch of differential gene expression in Brassica napus under low temperature stress (TOP20). Note: The larger the Rich Factor, the higher the degree of enrichment. Q-Value is the P-Value after multiple hypothesis test corrections, ranging from 0 to 1, the closer to zero, the more significant the enrichment. The figure is plotted using the Q-value from small to large to sort the top 20 paths.   Heat map of specific expression genes in the photosynthetically related significant enrichment pathway of Brassica napus L. Validaton of 17 differentially expressed genes by qRT-PCR