*2.7. Characterization of Anther Specific Gene OsACT*

To further find some candidate genes that may be involved in rice thermotolerance, real time PCR was also conducted on expression of some DEGs in anthers of 9311 with the same heat treatments as SDWG005. It was revealed that *OsACT* (LOC\_Os03g47860) showed quite different expression pattern between SDWG005 and 9311 under heat treatments (Figure 8a). Then, further analysis was conducted to understand the role of this gene in heat stress response. In silico study showed that this *OsACT* gene expressed specifically in anther and 5 DAPs' seeds (http://rice.plantbiology.msu.edu), and our analysis suggested that there was no detectable expression of *OsACT* in leaves of SDWG005 and 9311 at seedling stage either in control or high temperature condition (Data not shown). Coding region of *OsACT* was 1389 bp in length without intron, and the putative protein sequence contained 462 amino acids. The encoded sequence contained the HXXXD domain and DFGGG domain for BAHD family at C-terminal and N-terminal, respectively.

**Figure 8.** Expression and polymorphic analysis of *OsACT*. (**a**) Expression levels of *OsACT* in anthers of SDWG005 and 9311 under heat treatments by qRT-PCR. (**b**) Polymorphic analysis of WK-box in SDWG005, 9311, N22, and rice core collections with different thermotolerance. Pink indicates minor allele, gray indicates major allele; Relative seed setting rate data was from Zha et al. [20].

*OsACT* was further cloned from SDWG005 and 9311, and it was found that there were one and three SNPs in 5 up-stream and coding region of *OsACT* between them, respectively. What is more, all the three SNPs in coding region lead to change of amino acid, but neither in HXXXD domain nor in DFGGG domain. However, the SNP (T/C) in 5 up-stream region (−878 bp) located in the cis-element called WK-box (TTTTCCAC) [21]. Then, *OsACT* was cloned from 12 other lines with different thermotolerance ability, including N22 and 11 lines from rice core collections. As shown in Figure 8b, totally five haplotypes were detected for WK-box, including TCCACT, TCTACA, and CCTACT for thermotolerant lines, while CCCACT for 9311 and TCTACT for the other nine thermo-sensitive lines.
