Construction of Yeast One-hybrid Bait Reporter Vector for Screening the Binding Proteins of Cassava MeCWINV 1 Promoter

Cellwall invertase (CWIN) hydrolyzes sucrose into glucose and fructose irreversibly, playing key roles in carbohydrate partitioning and plant defence. MeCWINV1 is one of the CWINs in cassava, which contains several light-responsive elements and stress-responsive elements in promoter region. To analyze the regulatory function of MeCWINV1 in the cassava starch accumulation and stress defense response, a 865 bp MeCWINV1 promoter fragment was cloned and inserted into yeast one-hybrid bait vector to construct pCW1-AbAi vector, then was transformed to Y1HGold yeast strains to screen the binding proteins. It might provide a framework for further investigation on the regulation mechanism of MeCWINV1 gene in cassava.


Introduction
Soluble sugars, mostly hexoses and sucrose, which not only provide energy and carbon resources, but also act as signalling molecules in plant defence.Invertase (INV) hydrolyzes sucrose into glucose and fructose, thereby playing crucial roles in primary metabolism, plant development and defence response [1].In plant cells, invertases are found located in the cell wall, vacuole and cytosol, which are named as CWIN, VIN and CIN, respectively.CWIN and VIN are acid invertases with optimum pH 4.5-5.5, while CIN is neutral/alkaline invertases [2].CWINs serve as key metabolic enzymes in the carbohydrate supply of sink tissues and the regulation of source-sink transitions.Meanwhile CWINs are regulated by sugar-, hormone-, stress-related signals, and generate sugar-based signals via the modulation of sucrose/hexose ratios.Hence, several signals converge at the site of CWIN regulation.CWIN acts at an integration point of hormonal, sugar, defence and diurnal/circadian regulation [3].
Cassava has six CWIN genes MeCWINV1~6, these genes show different spatial and temporal patterns of expression during the cassava development.MeCWINV1 and MeCWINV3 had higher activity than other MeCWINVs in cassava leaves and tubers, which suggested MeCWINV1 and 3 were the key CWINs for catalyzing sucrose in the apoplastic space of cassava source and sink organs [4].In previous research we found that the MeCWINV1 gene promoter contains several light-responsive a Corresponding author: liujiao@itbb.org.cnelements and stress-responsive elements, which might be associated to the invertase gene function of cassava starch accumulation and stress defense [5].
In this study we cloned a 865 bp MeCWINV1 promoter fragment and constructed the yeast onehybrid bait vector pCW1-AbAi vector, and further transformed to Y1HGold yeast strains then determined the minimal inhibitory concentration of Aureobasidin A for Y1HGold[CW1/AbAi] strains.It can be used to screen the binding proteins of MeCWINV1 gene promoter and provide a framework to further investigate the regulation mechanism and signal pathways of MeCWINV1 gene.

Cloning and synthesizing pCW1-AbAi plasmid
The forward primer with Kpn I site (5'-GGGGTACCATGCGACTTGAAACTT-3') and the reverse primer with Sal I site (5'-ACGCGTCGACCTCTATTTTCCCTCT-3') was designed according to the known MeCWINV1 gene promoter sequences in the NCBI GenBank database (KC465190).The MeCWINV1 promoter region was amplificated from the pCW1-19T plasmid by PCR using the primers.The PCR product and pAbAi Y1H bait vector were digested with Kpn I and Sal I, then ligated at 16 overnight with T4 DNA ligase.The promoter was inserted into the pAbAi vector to generate a recombinant plasmid pCW1-AbAi, then was identified by PCR, restriction analysis, and sequencing.

Generating the bait-reporter yeast strains
Linearize 1 μg of pCW1-AbAi plasmid and p53-AbAi plasmid (positive control) by BstBI restriction enzyme.Transformed Y1HGold yeast strains with 1 μg of each of the linear plasmids using the protocol for the Yeastmaker Yeast Transformation System 2. Diluted each transformation reaction 1/10, 1/100, and 1/1000, then plated 100 μl from each dilution on SD/-Ura agar medium.After 3 days picked 5 colonies and analyzed by colony PCR using the Matchmaker Insert Check PCR Mix 1 (Clontech, Cat.No.630496).Used untransformed Y1HGold colonies as negative controls.Pick one colony for each confirmed bait, and one from the p53-AbAi control, and streak them onto SD/-Ura agar medium.After 3 days at 30 , store at 4 for up to 1 month.For long term storage, growed an overnight culture in YPDA broth, collect the cells by centrifugation, and resuspend them in 1 ml of freezing medium.Quick freezed, and stored at -70 .

Constructon and identification of pCW1-AbAi bait reporter vector
A 865 bp MeCWINV1 promoter fragment was isolated from the pCW1-19T plasmid (Fig. 1a) and inserted into the pAbAi vector to construct the yeast one-hybrid bait vector pCW1-AbAi.The recombinant plasmid pCW1-AbAi was digested by Kpn and Sal , approximate 865 bp fragments were obtained, which was consistent with MeCWINV1 promoters in length (Fig. 1b).Sequencing results showed that we obtained the recombinant pCW1-AbAi vector successfully.

Generating and identifying the bait-reporter yeast strains Y1HGold[CW1/AbAi]
The BstBI linearized pCW1-AbAi plasmid, p53-AbAi positive control plasmid were transformed into Y1HGold yeast strains and selected for transformants on SD/-Ura media.The plasmids confirmed to integrate correctly by using a colony PCR analysis.The primers in the Matchmaker Insert Check PCR Mix 1 were located in the AbA r gene and in the Y1Hgold genome, downstream of the URA3 locus.The positive control Y1HGold[p53-AbAi] amplified a region of ~1.4 kb (Fig. 2, line 4), each bait strain Y1HGold[CW1/AbAi] amplified a ~2 kb band containing 1.35 kb + insert size (865 bp) that encompassed the bait sequence and confirmed the presence of the integrated plasmid (Fig. 2, line 1-3).Thus the bait-reporter strain Y1HGold[CW1/AbAi] is generated by homologous integration into Y1Hgold.The integrated plasmids are very stable, overnight broth media culturing without URA3 selection will not result in loss of the integrant.Successful use of any yeast one-hybrid system depends upon no/low recognition of target sequence by endogenous yeast transcription factors.For this reason, it is critical to test the construct for AbA r expression before screening the library.The basal expression of the bait reporter stain in the absence of prey can vary, depended on the bait sequences cloned into pAbAi vector.If 1000 ng/ml Aureobasidin A (AbA) does not suppress AbA r in the absence of prey, the bait DNA sequence is likely being recognized by endogenous yeast transcription factors and therefore the sequence cannot be used for a yeast one hybrid screen.As recorded in the protocol, the positive control Y1HGold[p53-AbAi] has a minimal inhibitory concentration of 100 ng/ml AbA (Fig. 3).Our bait strain Y1HGold[CW1/AbAi] plated on SD/-Ura media with 60~80 ng/ml AbA had less clones compared to the control and no clones were appear when the concentration of AbA above100 ng/ml.Therefore, the minimal inhibitory concentration of AbA for our bait strain Y1HGold[CW1/AbAi] is 100 ng/ml (Fig. 3).For the follow-up library screening, we can use the minimal concentration of AbA, or a concentration that is slightly higher (by 50~100 ng/ml) to suppress the growth of the bait strain completely.