干旱脅迫下割手密基因cDNA—SCoT差異表達分析（英文）

吳凱朝 韋莉萍 徐 林 唐仕云 魏源文 黃誠梅 曹輝慶 羅海斌 蔣勝理 鄧智年 李楊瑞

摘要：【目的】通過分析割手密葉片在干旱脅迫下的基因表達譜，篩選獲得抗旱相關(guān)基因，為開展甘蔗抗旱性遺傳改良研究提供候選基因?！痉椒ā恳愿钍置蹽X83-10為材料，構(gòu)建正常澆灌（對照）及兩個干旱處理葉片總RNA混合池，使用cDNA-SCoT構(gòu)建割手密GX83-10伸長期應(yīng)答干旱脅迫的基因表達譜，篩選、分離轉(zhuǎn)錄衍生片段（TDFs）并進行測序，根據(jù)NCBI數(shù)據(jù)庫BLAST同源性檢索結(jié)果推測基因功能，并使用qRT-PCR對抗旱相關(guān)TDFs進行表達驗證分析?！窘Y(jié)果】成功獲得120個上調(diào)表達TDFs序列，其中53個TDFs序列與NCBI數(shù)據(jù)庫中已錄入的基因具有較高相似性，根據(jù)同源基因功能可分為10個類群：結(jié)合功能蛋白相關(guān)基因（20.75%）、新陳代謝相關(guān)基因（13.21%）、通信及信號轉(zhuǎn)導(dǎo)相關(guān)基因（13.21%）、轉(zhuǎn)錄調(diào)控因子相關(guān)基因（13.21%）、運輸途徑相關(guān)基因（11.32%）、環(huán)境互作相關(guān)基因（9.43%）、能量代謝相關(guān)基因（7.55%）、蛋白質(zhì)合成相關(guān)基因（3.77%）、防御相關(guān)基因（3.77%）及細胞成分生物合成相關(guān)基因（3.77%）。通過對運輸因子家族蛋白、RING/U-box超家族蛋白、22 kD干旱誘導(dǎo)蛋白、微管蛋白alpha-3鏈和質(zhì)膜H+-ATPase進行qRT-PCR分析，結(jié)果表明，這些基因在干旱脅迫下均呈不同程度的上調(diào)表達。【結(jié)論】干旱脅迫下，應(yīng)用cDNA-SCoT構(gòu)建割手密葉片基因表達譜篩選抗旱相關(guān)基因具有可行性。從割手密葉片中挖掘獲得的抗旱及水分有效利用相關(guān)基因，可為利用野生種質(zhì)資源開展甘蔗育種研究提供候選基因，改良甘蔗抗旱性，拓寬甘蔗育種基因庫。

關(guān)鍵詞： 割手密；cDNA-SCoT；轉(zhuǎn)錄衍生片段（TDFs）；基因；干旱脅迫

Abstract：【Objective】In order to provide candidate genes for improving drought resistance of sugarcane， the gene expression profiles in Saccharum spontaneum leaf under drought stress were analyzed for screening drought resistant genes. 【Method】S. spontaneum GX83-10 was used as material to design two treatments viz. normal watering（control，CK） and drought stress（T）， and their equal quantity mixed solutions of total RNA were constructed to establish gene expression profiles in response to drought stress at elongation stage of GX83-10 by using cDNA-SCoT differential display technology. The transcript derived fragments（TDFs） were screened and isolated for sequencing， followed by the BLAST homology searching in NCBI database to deduce gene function based on homological genes. Furthermore， the related genes of drought resistance were analyzed by real time fluorescence quantitative PCR（qRT-PCR）. 【Result】In this study， 120 up-regulated TDFs were successfully cloned and sequenced. Among them， 53 had higher similarity than others with the accessed genes in NCBI database， and these TDFs could be classified into 10 functional groups， including associated-functional protein related genes（20.75%）， metabolism related genes（13.21%）， communication and signal transduction related genes（13.21%）， transcription regulated factor related genes（13.21%）， transport pathway related genes（11.32%）， environmental interaction related genes（9.43%）， energy metabolism related genes（7.55%）， protein synthesis related genes（3.77%）， defense related genes（3.77%） and cell component biological synthesis related gene（3.77%）. Transport factor 2 family protein， RING/U-box superfamily protein， 22 kD drought-inducible protein， tubulin alpha-3 chain and plasma-membrane H+-ATPase were selected to conduct qRT-PCR analysis， and the results confirmed that all five genes were up-regulated in expressions at varying degrees under drought stress. 【Conclusion】Under drought stress， it is feasible to screen the drought resistance related genes in S. spontaneum from gene expression profiles constructed by applying cDNA-SCoT differential display technology. The drought resistance and water efficient utilization genes， which were excavated from S. spontaneum leaf， will provide more candidate genes for sugarcane breeding research using wild germplasm resources to improve sugarcane drought resistance and broaden the genetic library of sugarcane breeding.

Key words： Saccharum spontaneum； cDNA-SCoT； transcript derived fragments（TDFs）； gene； drought stress

CLC number： S566.1 Document code： A Article：2095-1191（2018）02-0201-07

0 Introduction

【Research significance】Saccharum spontaneum is the most important genetic basis for modern sugarcane varieties， and in these sugarcane varieties， 10%-25% chromosomes derived from S. spontaneum（Glaszmann et al.， 1989； DHont et al.， 1993）. S. spontaneum have many excellent characteristics such as drought， cold， barren and strong disease resistance and strong tillering， and it is the most valuable and the most used wild germplasm resource in sugarcane breeding. So screening important genes related to drought resistance in S. spontaneum will be of great significance for genetic improvement of su-garcane drought resistance， and even for other crops.【Research progress】With the development of plant transgenic technology， it will be a new way to improve the sugarcane variety by using the excellent gene of S. spontaneum. Previous studies had shown that 70 up-regulated genes were screened by using high-throughput transcriptome sequencing in S. spontaneum YN82-114 root under drought stress（Liu et al.， 2017）， 823 differentially expressed genes between S. officinarum and S. spontaneum were obtained by using gene chip technology and Solexa technology（Hou et al.， 2013）， these genes were involved in the regulation of multiple pathways. At present， two peroxidase（POD） allele sequences（SsPOD-1a and SsPOD-1b）（Hu et al.， 2015）， copper and zinc superoxide dismutas（SOD） gene（SsSOD-1a）sequence（Yao et al.， 2015）， four DREB2（SsDREB2-a，SsDREB2-f，SsDREB2-1 and SsDREB2-2） gene sequences（Yao et al.， 2016a， 2016b）， glutathione thiotransferase（GST） gene（SsGST） sequence（Xu et al.， 2015） and two catalase（CAT） gene（SsCAT-1c and SsCAT-1d） sequences（Liu et al.， 2014） were cloned from S. spontaneum. These excellent genes established the researching foundations for sugarcane resistance improvement. 【Research breakthrough point】At the bottleneck period of “noble” sugarcane bree-ding， using wild germplasm resources in sugarcane breeding is a feasible and effective way to improve the drought resistance of sugarcane. So， separating important drought resistant genes from S. officinarum wild germplasm and being used for genetic transformation research of sugarcane will be a parti-cularly important and urgent subject. At present， the drought resistant genes which were isolated and identified from S. officinarum wild germplasm and could be used for sugarcane breeding to help improve drought resistance of sugarcane have been less reported. Especially， the researches on gene screening from S. officinarum using cDNA-SCoT technology have not been reported. 【Solving problems】In this study， gene expression profiles of GX83-10 in response to drought stress at elongation stage were constructed using cDNA-SCoT differential display. The transcript derived fragments（TDFs） were screened and isolated for sequencing， then the related gene functions were deduced through BLAST homology searching in NCBI database. With these efforts，this study aims to excavat drought resistance related and water efficient utilization gene resources from S. officinarum wild germplasm of sugarcane to broaden the sugarcane breeding gene bank， and solve the pro-blem that the source of candidate genes for molecule genetic improvement of sugarcane resistance is narrowed down， which will lay the foundation for sugarcane breeding by using wild germplasm to improve the drought resistance of sugarcane.

1 Materials and methods

1. 1 Experimental materials

This experiment was carried out in greenhouse in 2014. S.spontaneum GX83-10， the drought and barren type， was used as tested material. The hydroponics rooting seedlings of GX83-10 were planted in black plastic buckets（35 cm of height and upper dia-meter， 28 cm of bottom diameter） on 5， September 2014. One seedling was planted in each bucket， and there were 40 buckets in total.

1. 2 Experimental design

The experiment designed normal watering treatment（CK） and drought stress treatment（T） by using completely randomized block arrangement. The GX83-10 seedlings of drought treatment at elongation stage were stopped to watering on 25， December， and its leaves were sampled at 10：00 a.m. after 4（T1）， 7（T2） and 11（T3） days and the +1 leaves of three plants were sampled from each time point. At the same time， the +1 leaves of three plants were sampled from CK. All the leaves were cut into segments and wiped off midrib， followed by treating with liquid nitrogen freezing and preserving at -85 ℃. The relative water content of soil was determined by using drying weighing method. The relative water content of soil for drought stress was 60.1%， 45.6% and 31.9% after stopping water for 4， 7 and 11 days respectively， which reached mild drought， moderate drought and severe drought levels（Zhu et al.， 2010）. And the relative water content of soil in CK reached about 80.0% for a suitable range of S. spontaneum growth at elongation stage. Variation of soil water content in all the buckets were tracked and determined by TZS-1 portable moisture meter throughout the experiment， and these measured data were used for deciding drought stress time and sampling time.

1. 3 Total RNA isolation， RNA mixed solution construction， and cDNA first strand synthesis

Total RNA of leaf was isolated using Trizol reagent and the method described by Wu et al.（2012）. The purification and concentration of total RNA were detected by ultraviolet spectrophotometer. Equal amount of total RNA in each drought time point（T1， T2 and T3） were mixed to construct sample mixture（T）， and the same amount of total RNA of control was mixed to construct sample solution（CK）. The cDNA first strand synthesis was conduc-ted by using AMV First Strand cDNA Kit of Fermentas Company， and operated according to the instructions. The 3.0 ?L of reverse transcriptase product was used for detecting the effects of cDNA first chain synthesis in 1.5% agarose gel electrophoresis.

1. 4 Amplification of SCoT-PCR and its pro-ducts detection

The SCoT-PCR amplification system was constructed according to the method described by Wu et al.（2013）. 1.0 ?L of amplified product was detected in 1.5% agarose gel electrophoresis， and the amplified products with good amplification and repeated results were used to construct cDNA-SCoT genes expression profiles by 7% non-denatured polyacryla-mide gels.

1. 5 Recovery， verification， cloning， sequencing and analysis of transcript derived fragments（TDFs）

TDFs recovery， re-amplification， purification， cloning， sequencing and sequence biologic analysis were conducted according to methods described by Wu et al.（2016）.

1. 6 Real-time PCR（qRT-PCR）

The relative expression of target TDFs in CK， T1， T2 and T3 cDNA were inspected by qRT-PCR， and beta-actin gene was used as referen-ce gene. Total RNA for qRT-PCR of each sample was isolated using Omini Plant RNA Kit DNase I（CW Biotech， Beijing， China）. The cDNA was synthesized using HiScript II Q RT SuperMix for qRT-PCR added gDNA_wiper（Vazyme Biotech， Nanjing， China）. Fluorescent quantitative PCR agent（AceQ qPCR SYBR Green Master Mix） was run on AnalytikJena qTOWERE2.2 qRT-PCR system（Germany） to detect relative gene expression level. Five up-regulated TDFs were selected for validation test and primers were designed according to the nucleotide sequences of each TDF. The relative expression of TDFs was calculated by 2-ΔΔCt.

2 Results and analysis

2. 1 Total RNA isolation， reverse-transcription and quality of PCR amplification

Based on the results of Fig.1， two clear and completing bands without trailing， 28S and 18S， were found in two total RNA samples of S. sponta-neum leaf， which indicated that the total RNA obtained was more complete， and no genomic DNA pollution and no degradation were observed. The determination results of two total RNA samples by ultraviolet spectrophotometer showed that OD260/OD230 ratio of CK and T treatments were 2.12 and 2.03 respectively， and their OD260/OD280 ratios were 1.89 and 1.94 respectively， indicating that the purity of total RNA was high and could be used for cDNA first chain synthesis. The results from Fig.2 presen-ted that， the first strand cDNA reverse transcribed was in diffused and uniform distribution， which showed that the reverse transcriptase reaction was full and the quality was fine， and it could meet the demand of cDNA-SCoT differential display analysis. The detected results of PCR product amplification by 1.5% agarose gel showed that the amplification strip was clear and the separation effects of bands were better（Fig.3）， which fitted for construction of cDNA-SCoT expression spectrum and screening of differential genes.

2. 2 Construction of gene expression profiles and screening of differential fragments using cDNA-SCoT under drought stress

Differential expression of cDNA-SCoT was an effective method for screening plant differential genes（Wu et al.， 2013）. After screening primers， gene expression profiles of 27 SCoT primers were constructed in this study， about 1300 bands were found and ranged from 100 to 2000 bp， and each primer got 19-52 bands amplified， which indicated that cDNA-SCoT was rich in polymorphism and was effective to isolate differential expression genes in S. spontaneum. The amplified results of primer S14， S15， S12， S13， S21， S22， S23， S24 and S19 were shown in Fig.4. The gene expression profiles with good reproducibility were analyzed， and the differential bands were screened for recycling. After recycling， re-amplification and purification， 276 differen-tial bands were successfully obtained， of which 152 up-regulated bands and 124 down-regulated bands were presented.

2. 3 Biological analysis and functional classification of differential fragments

The up-regulated TDFs reclaimed and purified were cloned and sequenced， and then their sequen-ces were analyzed and screened through comparing NCBI database. A total of 120 up-regulated TDFs sequences were successfully obtained， including 37 unknown TDFs， 30 unknown functional TDFs， and 53 known functional TDFs（Table 1）. They all had higher similarity with accessed genes in NCBI database than the others. The 53 known functional TDFs could be classified into 10 functional groups（Fig.5）， which included associated-functional protein related genes（20.75%）， metabolism related genes（13.21%）， communication and signal transduction related genes（13.21%）， transcription regulated factor related genes（13.21%）， transport pathway related genes（11.32%）， environmental interaction related genes（9.43%）， ener-gy metabolism related genes（7.55%）， protein synthesis related genes（3.77%）， defense related genes（3.77%）， cell component biological synthesis related gene（3.77%）. In this study， bidirectional sugar transporter， sucrose transporter（SUT）， tubulin alpha， drought-inducible protein， IAA-auxin-responsive protein， plasma-membrane H+-ATPase， fucose synthase and dehydrin（DHN）， the important regulatory factors in plant anti-reverse physiology， were up-regulated expression in S. spontaneum leaves under drought stress， which indicated that S. spontaneum colud positively respond to drought stress and actively defense to preserve the plant normal physiological function.

2. 4 Expression analysis of up-regulated genes

qRT-RCR analysis is a very effective method to deduce the initial quantity of target genes. The up-regulated gene screened might be closely related to drought toleran-ce of S. spontaneum， so five up-re-gulated TDFs viz.， transport factor 2 family protein， RING/U-box superfamily protein， 22 kD drought-inducible protein， tubulin alpha-3 chain and plasma-membrane H+-ATPase by cDNA-SCoT analysis in this study were selected for real-time quantitative analysis. The results showed that， the up-regulated level of transport factor 2 family protein， RING/U-box superfamily protein and plasma-membrane H+-ATPase genes reached the highest level under the moderate drought stress， tubulin alpha-3 chain gene presented a high up-regulated expression under severe drought stress， and 22 kD drought-inducible protein gene had the highest up-regulated expression under modera-te and severe drought stress， which proved that all the five genes were induced to up-regulate by drought stress， and they might play a regulatory role in response to drought stress（Fig.6）.

3 Discussion

S. spontaneum had been used in sugarcane cross breeding， and it had formed parent system with Chinese characteristics. The corresponding hybrids were obtained and applied to sugarcane breeding by interspecific hybridization and molecular marker identification（Li， 2010）. These hybrids by interspecific hybridization were of great significance for promoting the cross breeding of sugarcane resistance in China. For example， GT21 and GT29， they had the drought resistance and cold resistance of S. spontaneum through the traditional cross breeding. In the molecular genetic improvement research of sugarcane drought resistance， the source of candidate genes is too narrow and new breakthroughs have not been achieved. Therefore， it is still the goal of our efforts to obtain a new genetically modified sugarcane varie-ties（materials） to resist drought stress. And it is also very important and urgent to exploit candidate genes suitable for sugarcane genetic transformation from the wild germplasm resources， such as S. spontaneum. In this study， the TDFs which were related to drought stress were isolated from S. spontaneum GX83-10 leaves， and their expression under water stress was preliminarily identified， which would provide a scientific basis for further functional identification of these related genes， provide more candidate genes for utilizing wild germplasm resources to improve sugarcane drought resistance in sugarcane breeding， and broaden the gene bank of sugarcane breeding. At present， the functions of some drought resistant genes had been identified in the research of S. spontaneum genes exploited and utilized， but the genetic improvement with candidate genes of S. spontaneum had not yet been found. Therefore， the research field of S. spontaneum genes exploited and utilized is still at an initial stage， and it will be a potential field， too.

Since the whole genome of sugarcane has not been published， it is very important to select candidate genes from wild germplasm resources to improve the drought resistance of sugarcane. In this study， cDNA-SCoT differential display analysis was used for screening the genes related to drought resistance in S. spontaneum， and a number of meaningful genetic information were obtained. The related TDFs， sucrose transporter and H+-ATPase were similar to the screening results in GT11（Wu et al.， 2016）. Equally， IAA-auxin-responsive protein had also been found in leaf transcriptome of S. spontaneum under drought stress（another publication）. Furthermore， fucose synthase and dehydrin genes were screened， they were proved to be drought resistant genes in plants and their transgenic plants to enhance the drought resistance of the plants（Zhang et al.， 2016； Nie et al.， 2017）. These genes were likely to play an important role in drought tolerance regulation， which was worth further research and identification to provide scientific basis for genetic improvement of drought resistant sugarcane in the future.

4 Conclusion

In a conclusion， the cDNA-SCoT differential display technology could successfully separated the related genes from S. spontaneum under drought stress， and some genes related to drought resistance were preliminarily screened， which provided rich information for further understanding the molecular mechanism in S. spontaneum response to drought. The drought resistance and water efficient utilization genes that excavated from S. spontaneum would provide more candidate genes for sugarcane breeding research by using wild germplasm resources to improve sugarcane drought resistance， and broaden the genetic library of sugarcane breeding.

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（責(zé)任編輯 陳德元）